Amino acid sequences directed against multitarget scavenger receptors and polypeptides

ABSTRACT

The present invention relates to amino acid sequences that are directed against (as defined herein) multitarget scavenger receptors such as e.g. Lox-1, RAGE, CD36, SR-A1, SR-B1, galectin-1, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences (also referred to herein as “amino acid sequences of the invention”, “compounds of the invention”, and “polypeptides of the invention”, respectively).

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 ofinternational application PCT/EP2009/059424, filed Jul. 22, 2009, whichwas published under PCT Article 21(2) in English, and claims the benefitunder 35 U.S.C. §119(e) of U.S. provisional application Ser. No.61/082,614, filed Jul. 22, 2008, the disclosures of which areincorporated by reference herein in their entireties.

The present invention relates to amino acid sequences that are directedagainst (as defined herein) multitarget scavenger receptors such as e.g.Lox-1, RAGE, CD36, SR-A1, SR-B1, galectin-1, as well as to compounds orconstructs, and in particular proteins and polypeptides, that compriseor essentially consist of one or more such amino acid sequences (alsoreferred to herein as “amino acid sequences of the invention”,“compounds of the invention”, and “polypeptides of the invention”,respectively).

The invention also relates to nucleic acids encoding such amino acidsequences and polypeptides (also referred to herein as “nucleic acids ofthe invention” or “nucleotide sequences of the invention”); to methodsfor preparing such amino acid sequences and polypeptides; to host cellsexpressing or capable of expressing such amino acid sequences orpolypeptides; to compositions, and in particular to pharmaceuticalcompositions, that comprise such amino acid sequences, polypeptides,nucleic acids and/or host cells; and to uses of such amino acidsequences or polypeptides, nucleic acids, host cells and/orcompositions, in particular for prophylactic, therapeutic or diagnosticpurposes, such as the prophylactic, therapeutic or diagnostic purposesmentioned herein.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

The polypeptides and compositions of the present invention can generallye.g. be used to modulate, and in particular inhibit and/or prevent,binding of Advanced Glycation End products (hereinafter “AGE”) ormodified LDL, polyribonucleotides, polysaccharides (including LPS), andanionic phospholipids “ligands to multitarget scavenger receptors”), tomultitarget scavenger receptors, and thus to modulate, and in particularinhibit or prevent, the signalling that is mediated by said ligandsand/or said receptors, to modulate the biological pathways in which saidligands and/or said receptors are involved, and/or to modulate thebiological mechanisms, responses and effects associated with suchsignalling or these pathways.

As such, the polypeptides and compositions of the present invention canbe used for the prevention and treatment (as defined herein) ofamyloidosis, amyloidosis such as Alzheimer, arteriosclerosis, diabetesand arthritis. Generally, the diseases or disorders of the invention canbe defined as diseases and disorders that can be prevented and/ortreated, respectively, by suitably administering to a subject in needthereof (i.e. having the disease or disorder or at least one symptomthereof and/or at risk of attracting or developing the disease ordisorder) of either a polypeptide or composition of the invention (andin particular, of a pharmaceutically active amount thereof) and/or of aknown active principle active against multiscavenger receptors or abiological pathway or mechanism in which multiscavenger receptors isinvolved (and in particular, of a pharmaceutically active amountthereof).

In particular, the polypeptides and compositions of the presentinvention can be used for the prevention and treatment ofarteriosclerosis, diabetes, and arthritis which are characterized byexcessive and/or unwanted signalling mediated by multiscavengerreceptors or by the pathway(s) in which multiscavenger receptors areinvolved such as e.g. inflammatory processes. Examples of such undesiredsignalling will again be clear to the skilled person based on thedisclosure herein.

Thus, without being limited thereto, the amino acid sequences andpolypeptides of the invention can for example be used to prevent and/orto treat all diseases and disorders that are currently being preventedor treated with active principles that can modulate multiscavengerreceptors-mediated signalling, such as those mentioned in the prior art.It is also envisaged that the polypeptides of the invention can be usedto prevent and/or to treat all diseases and disorders for whichtreatment with such active principles is currently being developed, hasbeen proposed, or will be proposed or developed in future. In addition,it is envisaged that, because of their favourable properties as furtherdescribed herein, the polypeptides of the present invention may be usedfor the prevention and treatment of other diseases and disorders thanthose for which these known active principles are being used or will beproposed or developed; and/or that the polypeptides of the presentinvention may provide new methods and regimens for treating the diseasesand disorders described herein.

Other applications and uses of the amino acid sequences and polypeptidesof the invention will become clear to the skilled person from thefurther disclosure herein.

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the diagnosis, prevention and/or treatment of amyloidosis,amyloidosis such as Alzheimer, arteriosclerosis, diabetes and arthritisand of the further diseases and disorders mentioned herein or suggestedby the prior art for multitarget scavenger as disclosed herein; and toprovide methods for the diagnosis, prevention and/or treatment of suchdiseases and disorders that involve the administration and/or use ofsuch agents and compositions.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions and/or methods that havecertain advantages compared to the agents, compositions and/or methodsthat are currently used and/or known in the art. These advantages willbecome clear from the further description below.

More in particular, it is an object of the invention to providetherapeutic proteins that can be used as pharmacologically activeagents, as well as compositions comprising the same, for the diagnosis,prevention and/or treatment of amyloidosis such as Alzheimer,arteriosclerosis, diabetes and arthritis and of the further diseases anddisorders mentioned herein; and to provide methods for the diagnosis,prevention and/or treatment of such diseases and disorders that involvethe administration and/or the use of such therapeutic proteins andcompositions.

Accordingly, it is a specific object of the present invention to provideamino acid sequences that are directed against (as defined herein)multiscavenger receptors, in particular against multiscavenger receptorsfrom a warm-blooded animal, more in particular against multiscavengerreceptors from a mammal, and especially against human multiscavengerreceptors; and to provide proteins and polypeptides comprising oressentially consisting of at least one such amino acid sequence.

In particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat are suitable for prophylactic, therapeutic and/or diagnostic use ina warm-blooded animal, and in particular in a mammal, and more inparticular in a human being.

More in particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat can be used for the prevention, treatment, alleviation and/ordiagnosis of one or more diseases, disorders or conditions associatedwith multiscavenger receptors and/or mediated by multiscavengerreceptors (such as the diseases, disorders and conditions mentionedherein) in a warm-blooded animal, in particular in a mammal, and more inparticular in a human being.

It is also a specific object of the invention to provide such amino acidsequences and such proteins and/or polypeptides that can be used in thepreparation of pharmaceutical or veterinary compositions for theprevention and/or treatment of one or more diseases, disorders orconditions associated with and/or mediated by multiscavenger receptors(such as the diseases, disorders and conditions mentioned herein) in awarm-blooded animal, in particular in a mammal, and more in particularin a human being.

In the invention, generally, these objects are achieved by the use ofthe amino acid sequences, proteins, polypeptides and compositions thatare described herein.

In general, the invention provides amino acid sequences that aredirected against (as defined herein) and/or can specifically bind (asdefined herein) to multiscavenger receptors; as well as compounds andconstructs, and in particular proteins and polypeptides, that compriseat least one such amino acid sequence.

More in particular, the invention provides amino acid sequences that canbind to multiscavenger receptors with an affinity (suitably measuredand/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value(actual or apparent), a k_(on)-rate and/or a k_(off)-rate, oralternatively as an IC₅₀ value, as further described herein) that is asdefined herein; as well as compounds and constructs, and in particularproteins and polypeptides, that comprise at least one such amino acidsequence.

In particular, amino acid sequences and polypeptides of the inventionare preferably such that they:

-   -   bind to multiscavenger receptors with a dissociation constant        (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably        10⁻⁷ to 10⁻¹² moles/liter or less and more preferably 10⁻⁸ to        10⁻¹² moles/liter (i.e. with an association constant (K_(A)) of        10⁵ to 10¹² liter/moles or more, and preferably 10⁷ to 10¹²        liter/moles or more and more preferably 10⁸ to 10¹²        liter/moles);        and/or such that they:    -   bind to multiscavenger receptors with a k_(on)-rate of between        10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹        and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷        M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to multiscavenger receptors with a k_(off) rate between 1        s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶        s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is preferably such that it will bind to multiscavenger receptors with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 500 pM.

Some preferred IC50 values for binding of the amino acid sequences orpolypeptides of the invention to multiscavenger receptors will becomeclear from the further description and examples herein.

For binding to multiscavenger receptors, an amino acid sequence of theinvention will usually contain within its amino acid sequence one ormore amino acid residues or one or more stretches of amino acid residues(i.e. with each “stretch” comprising two or amino acid residues that areadjacent to each other or in close proximity to each other, i.e. in theprimary or tertiary structure of the amino acid sequence) via which theamino acid sequence of the invention can bind to multiscavengerreceptors, which amino acid residues or stretches of amino acid residuesthus form the “site” for binding to multiscavenger receptors (alsoreferred to herein as the “antigen binding site”).

The amino acid sequences provided by the invention are preferably inessentially isolated form (as defined herein), or form part of a proteinor polypeptide of the invention (as defined herein), which may compriseor essentially consist of one or more amino acid sequences of theinvention and which may optionally further comprise one or more furtheramino acid sequences (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or more aminoacid sequences of the invention may be used as a binding unit in such aprotein or polypeptide, which may optionally contain one or more furtheramino acid sequences that can serve as a binding unit (i.e. against oneor more other targets than multiscavenger receptors), so as to provide amonovalent, multivalent or multispecific polypeptide of the invention,respectively, all as described herein. Such a protein or polypeptide mayalso be in essentially isolated form (as defined herein).

The amino acid sequences and polypeptides of the invention as suchpreferably essentially consist of a single amino acid chain that is notlinked via disulphide bridges to any other amino acid sequence or chain(but that may or may not contain one or more intramolecular disulphidebridges. For example, it is known that Nanobodies—as described hereinmay sometimes contain a disulphide bridge between CDR3 and CDR1 or FR2).However, it should be noted that one or more amino acid sequences of theinvention may be linked to each other and/or to other amino acidsequences (e.g. via disulphide bridges) to provide peptide constructsthat may also be useful in the invention (for example Fab′ fragments,F(ab′)₂ fragments, ScFv constructs, “diabodies” and other multispecificconstructs. Reference is for example made to the review by Holliger andHudson, Nat Biotechnol. 2005 September; 23(9):1126-36).

Generally, when an amino acid sequence of the invention (or a compound,construct or polypeptide comprising the same) is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is preferably either anamino acid sequence that does not occur naturally in said subject; or,when it does occur naturally in said subject, in essentially isolatedform (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use,the amino acid sequences of the invention (as well as compounds,constructs and polypeptides comprising the same) are preferably directedagainst human multiscavenger receptors; whereas for veterinary purposes,the amino acid sequences and polypeptides of the invention arepreferably directed against multiscavenger receptors from the species tobe treated, or at least cross-reactive with multiscavenger receptorsfrom the species to be treated. Furthermore, an amino acid sequence ofthe invention may optionally, and in addition to the at least onebinding site for binding against multiscavenger receptors, contain oneor more further binding sites for binding against other antigens,proteins or targets. The efficacy of the amino acid sequences andpolypeptides of the invention, and of compositions comprising the same,can be tested using any suitable in vitro assay, cell-based assay, invivo assay and/or animal model known per se, or any combination thereof,depending on the specific disease or disorder involved. Suitable assaysand animal models will be clear to the skilled person, and for exampleinclude the In vitro inhibition of transthyretin aggregate-inducedcytotoxicity by full and peptide derived forms of the soluble receptorfor advanced glycation end products (RAGE)—(see FEBS Letters, Volume580, Issue 14, Pages 3451-3456 F. Monteiro, I. Cardoso, M. Sousa, M.Saraiva), as well as the assays and animal models used in theexperimental part below and in the prior art cited herein.

Also, according to the invention, amino acid sequences and polypeptidesthat are directed against multiscavenger receptors from a first speciesof warm-blooded animal may or may not show cross-reactivity withmultiscavenger receptors from one or more other species of warm-bloodedanimal. For example, amino acid sequences and polypeptides directedagainst human multiscavenger receptors may or may not show crossreactivity with multiscavenger receptors from one or more other speciesof primates (such as, without limitation, monkeys from the genus Macaca(such as, and in particular, cynomologus monkeys (Macaca fascicularis)and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus))and/or with multiscavenger receptors from one or more species of animalsthat are often used in animal models for diseases (for example mouse,rat, rabbit, pig or dog), and in particular in animal models fordiseases and disorders associated with multiscavenger receptors (such asthe species and animal models mentioned herein). In this respect, itwill be clear to the skilled person that such cross-reactivity, whenpresent, may have advantages from a drug development point of view,since it allows the amino acid sequences and polypeptides against humanmultiscavenger receptors to be tested in such disease models.

More generally, amino acid sequences and polypeptides of the inventionthat are cross-reactive with multiscavenger receptors from multiplespecies of mammal will usually be advantageous for use in veterinaryapplications, since it will allow the same amino acid sequence orpolypeptide to be used across multiple species. Thus, it is alsoencompassed within the scope of the invention that amino acid sequencesand polypeptides directed against multiscavenger receptors from onespecies of animal (such as amino acid sequences and polypeptides againsthuman multiscavenger receptors) can be used in the treatment of anotherspecies of animal, as long as the use of the amino acid sequences and/orpolypeptides provide the desired effects in the species to be treated.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) ofmultiscavenger receptors against which the amino acid sequences andpolypeptides of the invention are directed. For example, the amino acidsequences and polypeptides may or may not be directed against an“interaction site” (as defined herein). However, it is generally assumedand preferred that the amino acid sequences and polypeptides of theinvention are preferably directed against an interaction site (asdefined herein).

As further described herein, a polypeptide of the invention may containtwo or more amino acid sequences of the invention that are directedagainst multiscavenger receptors. Generally, such polypeptides will bindto multiscavenger receptors with increased avidity compared to a singleamino acid sequence of the invention. Such a polypeptide may for examplecomprise two amino acid sequences of the invention that are directedagainst the same antigenic determinant, epitope, part, domain, subunitor confirmation (where applicable) of multiscavenger receptors (whichmay or may not be an interaction site); or comprise at least one “first”amino acid sequence of the invention that is directed against a firstsame antigenic determinant, epitope, part, domain, subunit orconfirmation (where applicable) of multiscavenger receptors (which mayor may not be an interaction site); and at least one “second” amino acidsequence of the invention that is directed against a second antigenicdeterminant, epitope, part, domain, subunit or confirmation (whereapplicable) different from the first (and which again may or may not bean interaction site). Preferably, in such “biparatopic” polypeptides ofthe invention, at least one amino acid sequence of the invention isdirected against an interaction site (as defined herein), although theinvention in its broadest sense is not limited thereto.

Also, when the target is part of a binding pair (for example, areceptor-ligand binding pair), the amino acid sequences and polypeptidesmay be such that they compete with the cognate binding partner (e.g. theligand, receptor or other binding partner, as applicable) for binding tothe target, and/or such that they (fully or partially) neutralizebinding of the binding partner to the target.

It is also within the scope of the invention that, where applicable, anamino acid sequence of the invention can bind to two or more antigenicdeterminants, epitopes, parts, domains, subunits or confirmations ofmultiscavenger receptors. In such a case, the antigenic determinants,epitopes, parts, domains or subunits of multiscavenger receptors towhich the amino acid sequences and/or polypeptides of the invention bindmay be essentially the same (for example, if multiscavenger receptorscontains repeated structural motifs or occurs in a multimeric form) ormay be different (and in the latter case, the amino acid sequences andpolypeptides of the invention may bind to such different antigenicdeterminants, epitopes, parts, domains, subunits of multiscavengerreceptors with an affinity and/or specificity which may be the same ordifferent). Also, for example, when multiscavenger receptors exists inan activated conformation and in an inactive conformation, the aminoacid sequences and polypeptides of the invention may bind to either oneof these confirmation, or may bind to both these confirmations (i.e.with an affinity and/or specificity which may be the same or different).Also, for example, the amino acid sequences and polypeptides of theinvention may bind to a conformation of multiscavenger receptors inwhich it is bound to a pertinent ligand, may bind to a conformation ofmultiscavenger receptors in which it not bound to a pertinent ligand, ormay bind to both such conformations (again with an affinity and/orspecificity which may be the same or different).

It is also expected that the amino acid sequences and polypeptides ofthe invention will generally bind to all naturally occurring orsynthetic analogs, variants, mutants, alleles, parts and fragments ofmultiscavenger receptors; or at least to those analogs, variants,mutants, alleles, parts and fragments of multiscavenger receptors thatcontain one or more antigenic determinants or epitopes that areessentially the same as the antigenic determinant(s) or epitope(s) towhich the amino acid sequences and polypeptides of the invention bind inmultiscavenger receptors (e.g. in wild-type multiscavenger receptors).Again, in such a case, the amino acid sequences and polypeptides of theinvention may bind to such analogs, variants, mutants, alleles, partsand fragments with an affinity and/or specificity that are the same as,or that are different from (i.e. higher than or lower than), theaffinity and specificity with which the amino acid sequences of theinvention bind to (wild-type) multiscavenger receptors. It is alsoincluded within the scope of the invention that the amino acid sequencesand polypeptides of the invention bind to some analogs, variants,mutants, alleles, parts and fragments of multiscavenger receptors, butnot to others.

When multiscavenger receptors exists in a monomeric form and in one ormore multimeric forms, it is within the scope of the invention that theamino acid sequences and polypeptides of the invention only bind tomultiscavenger receptors in monomeric form, only bind to multiscavengerreceptors in multimeric form, or bind to both the monomeric and themultimeric form. Again, in such a case, the amino acid sequences andpolypeptides of the invention may bind to the monomeric form with anaffinity and/or specificity that are the same as, or that are differentfrom (i.e. higher than or lower than), the affinity and specificity withwhich the amino acid sequences of the invention bind to the multimericform.

Also, when multiscavenger receptors can associate with other proteins orpolypeptides to form protein complexes (e.g. with multiple subunits), itis within the scope of the invention that the amino acid sequences andpolypeptides of the invention bind to multiscavenger receptors in itsnon-associated state, bind to multiscavenger receptors in its associatedstate, or bind to both. In all these cases, the amino acid sequences andpolypeptides of the invention may bind to such multimers or associatedprotein complexes with an affinity and/or specificity that may be thesame as or different from (i.e. higher than or lower than) the affinityand/or specificity with which the amino acid sequences and polypeptidesof the invention bind to multiscavenger receptors in its monomeric andnon-associated state.

Also, as will be clear to the skilled person, proteins or polypeptidesthat contain two or more amino acid sequences directed againstmultiscavenger receptors may bind with higher avidity to multiscavengerreceptors than the corresponding monomeric amino acid sequence(s). Forexample, and without limitation, proteins or polypeptides that containtwo or more amino acid sequences directed against different epitopes ofmultiscavenger receptors may (and usually will) bind with higher aviditythan each of the different monomers, and proteins or polypeptides thatcontain two or more amino acid sequences directed against multiscavengerreceptors may (and usually will) bind also with higher avidity to amultimer of multiscavenger receptors.

Generally, amino acid sequences and polypeptides of the invention willat least bind to those forms of multiscavenger receptors (includingmonomeric, multimeric and associated forms) that are the most relevantfrom a biological and/or therapeutic point of view, as will be clear tothe skilled person.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the amino acidsequences and polypeptides of the invention, and/or to use proteins orpolypeptides comprising or essentially consisting of one or more of suchparts, fragments, analogs, mutants, variants, alleles and/orderivatives, as long as these are suitable for the uses envisagedherein. Such parts, fragments, analogs, mutants, variants, allelesand/or derivatives will usually contain (at least part of) a functionalantigen-binding site for binding against multiscavenger receptors; andmore preferably will be capable of specific binding to multiscavengerreceptors, and even more preferably capable of binding to multiscavengerreceptors with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein. Some non-limitingexamples of such parts, fragments, analogs, mutants, variants, alleles,derivatives, proteins and/or polypeptides will become clear from thefurther description herein. Additional fragments or polypeptides of theinvention may also be provided by suitably combining (i.e. by linking orgenetic fusion) one or more (smaller) parts or fragments as describedherein.

In one specific, but non-limiting aspect of the invention, which will befurther described herein, such analogs, mutants, variants, alleles,derivatives have an increased half-life in serum (as further describedherein) compared to the amino acid sequence from which they have beenderived. For example, an amino acid sequence of the invention may belinked (chemically or otherwise) to one or more groups or moieties thatextend the half-life (such as PEG), so as to provide a derivative of anamino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of theinvention may be an amino acid sequence that comprises an immunoglobulinfold or may be an amino acid sequence that, under suitable conditions(such as physiological conditions) is capable of forming animmunoglobulin fold (i.e. by folding). Reference is inter alia made tothe review by Halaby et al., J. (1999) Protein Eng. 12, 563-71.Preferably, when properly folded so as to form an immunoglobulin fold,such an amino acid sequence is capable of specific binding (as definedherein) to multiscavenger receptors; and more preferably capable ofbinding to multiscavenger receptors with an affinity (suitably measuredand/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value(actual or apparent), a k_(on)-rate and/or a k_(off)-rate, oralternatively as an IC₅₀ value, as further described herein) that is asdefined herein. Also, parts, fragments, analogs, mutants, variants,alleles and/or derivatives of such amino acid sequences are preferablysuch that they comprise an immunoglobulin fold or are capable forforming, under suitable conditions, an immunoglobulin fold.

In particular, but without limitation, the amino acid sequences of theinvention may be amino acid sequences that essentially consist of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively); or any suitablefragment of such an amino acid sequence (which will then usually containat least some of the amino acid residues that form at least one of theCDR's, as further described herein).

The amino acid sequences of the invention may in particular be animmunoglobulin sequence or a suitable fragment thereof, and more inparticular be an immunoglobulin variable domain sequence or a suitablefragment thereof, such as light chain variable domain sequence (e.g. aV_(L)-sequence) or a suitable fragment thereof; or a heavy chainvariable domain sequence (e.g. a V_(H)-sequence) or a suitable fragmentthereof. When the amino acid sequence of the invention is a heavy chainvariable domain sequence, it may be a heavy chain variable domainsequence that is derived from a conventional four-chain antibody (suchas, without limitation, a V_(H) sequence that is derived from a humanantibody) or be a so-called V_(HH)-sequence (as defined herein) that isderived from a so-called “heavy chain antibody” (as defined herein).

However, it should be noted that the invention is not limited as to theorigin of the amino acid sequence of the invention (or of the nucleotidesequence of the invention used to express it), nor as to the way thatthe amino acid sequence or nucleotide sequence of the invention is (orhas been) generated or obtained. Thus, the amino acid sequences of theinvention may be naturally occurring amino acid sequences (from anysuitable species) or synthetic or semi-synthetic amino acid sequences.In a specific but non-limiting aspect of the invention, the amino acidsequence is a naturally occurring immunoglobulin sequence (from anysuitable species) or a synthetic or semi-synthetic immunoglobulinsequence, including but not limited to “humanized” (as defined herein)immunoglobulin sequences (such as partially or fully humanized mouse orrabbit immunoglobulin sequences, and in particular partially or fullyhumanized V_(HH) sequences or Nanobodies), “camelized” (as definedherein) immunoglobulin sequences, as well as immunoglobulin sequencesthat have been obtained by techniques such as affinity maturation (forexample, starting from synthetic, random or naturally occurringimmunoglobulin sequences), CDR grafting, veneering, combining fragmentsderived from different immunoglobulin sequences, PCR assembly usingoverlapping primers, and similar techniques for engineeringimmunoglobulin sequences well known to the skilled person; or anysuitable combination of any of the foregoing. Reference is for examplemade to the standard handbooks, as well as to the further descriptionand prior art mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturallyoccurring nucleotide sequences or synthetic or semi-synthetic sequences,and may for example be sequences that are isolated by PCR from asuitable naturally occurring template (e.g. DNA or RNA isolated from acell), nucleotide sequences that have been isolated from a library (andin particular, an expression library), nucleotide sequences that havebeen prepared by introducing mutations into a naturally occurringnucleotide sequence (using any suitable technique known per se, such asmismatch PCR), nucleotide sequence that have been prepared by PCR usingoverlapping primers, or nucleotide sequences that have been preparedusing techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular be a domainantibody (or an amino acid sequence that is suitable for use as a domainantibody), a single domain antibody (or an amino acid sequence that issuitable for use as a single domain antibody), a “dAb” (or an amino acidsequence that is suitable for use as a dAb) or a Nanobody™ (as definedherein, and including but not limited to a V_(HH) sequence); othersingle variable domains, or any suitable fragment of any one thereof.For a general description of (single) domain antibodies, reference isalso made to the prior art cited above, as well as to EP 0 368 684. Forthe term “dAb's”, reference is for example made to Ward et al. (Nature1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol.,2003, 21(11):484-490; as well as to for example WO 06/030220, WO06/003388 and other published patent applications of Domantis Ltd. Itshould also be noted that, although less preferred in the context of thepresent invention because they are not of mammalian origin, singledomain antibodies or single variable domains can be derived from certainspecies of shark (for example, the so-called “IgNAR domains”, see forexample WO 05/18629).

In particular, the amino acid sequence of the invention may be aNanobody® (as defined herein) or a suitable fragment thereof. [Note:Nanobody®, Nanobodies® and Nanoclone® are registered trademarks ofAblynx N.V.] Such Nanobodies directed against multiscavenger receptorswill also be referred to herein as “Nanobodies of the invention”.

For a general description of Nanobodies, reference is made to thefurther description below, as well as to the prior art cited herein. Inthis respect, it should however be noted that this description and theprior art mainly described Nanobodies of the so-called “V_(H)3 class”(i.e. Nanobodies with a high degree of sequence homology to humangermline sequences of the V_(H)3 class such as DP-47, DP-51 or DP-29),which Nanobodies form a preferred aspect of this invention. It shouldhowever be noted that the invention in its broadest sense generallycovers any type of Nanobody directed against multiscavenger receptors,and for example also covers the Nanobodies belonging to the so-called“V_(H)4 class” (i.e. Nanobodies with a high degree of sequence homologyto human germline sequences of the V_(H)4 class such as DP-78), as forexample described in WO 07/118,670.

Generally, Nanobodies (in particular V_(HH) sequences and partiallyhumanized Nanobodies) can in particular be characterized by the presenceof one or more “Hallmark residues” (as described herein) in one or moreof the framework sequences (again as further described herein).

Thus, generally, a Nanobody can be defined as an amino acid sequencewith the (general) structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which one or more of the Hallmark residuesare as further defined herein.

In particular, a Nanobody can be an amino acid sequence with the(general) structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which the framework sequences are as furtherdefined herein.

More in particular, a Nanobody can be an amino acid sequence with the(general) structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which. CDR1 to CDR3 refer to the complementarity determining regions1 to 3, respectively, and in which:

-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    B-2 below;    and in which:-   ii) said amino acid sequence has at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 1 to    22, in which for the purposes of determining the degree of amino    acid identity, the amino acid residues that form the CDR sequences    (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are    disregarded.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Thus, the invention also relates to such Nanobodies that can bind to (asdefined herein) and/or are directed against multiscavenger receptors, tosuitable fragments thereof, as well as to polypeptides that comprise oressentially consist of one or more of such Nanobodies and/or suitablefragments.

SEQ ID NO's: 308 to 333 (see Table A-1) give the amino acid sequences ofa number of V_(HH) sequences that have been raised againstmultiscavenger receptors.

TABLE A-1 Preferred VHH sequences or Nanobody sequences(also referred herein as a sequence with aparticular name or SEQ ID NO: X, wherein Xis a number referring to the relevant amino acid sequence): SEQ IDNO: X, Tar- wherein Name get X = Amino acid sequence >99B5 RAGE 308EVQLVESGGGLVQAGDSLRLSCIASGRTFT MGWERQAPGKEREEVAAISWSGGRTYYADSVKGRFTISRENAKNTVYLQMNSLKPED TAVYCCATENLASSGSAYSDDRYNACGQGTQVTVSS >99C6 RAGE 309 EVQLVESGGEVVQPGGSLRLSCAASGFTFDDRAIGWFRQAPGKEREGVACSANNDNR AFYEDSVKGRFAVSRDNAKNTVYLQMNSLKPEDTAVYYCATRCAAGRVNLYYGMD YWGKGTLVTVSS >99D3 RAGE 310EVQLVESGGGLVQPGGSLRLSCAASGFTL GNYAIGWFRQAPGKEREGVSCVDRDGGSTYYLDSVTGRFTTSRDDAENTVYLQMNSL IPDDTAVYYCATRLYGCSGYGRDYADWGQGTQVTVSS >108D11 RAGE 311 EVQLVESGGGLVQAGGSLRLSCAVSGRTFSTDAFGWFRQAPGKEREFVSAMRWNGSS SYYADLVKGRFTISRDNAKNTVYLLMNSLKPEDTAVYYCTAGKRYGYYDYWGQGTQ VTVSS >108D3 RAGE 312EVQLVESGGGLVQAGGSLRLSCAASGRTF SNYSMGWFRQAPGKEREFVATISWSGALTHYTDSVKGRFTISRDNAKNTVYLQMNSLK PEDTAVYYCAASDSDYGNKYDYWGQGT QVTVSS >108E3RAGE 313 EVQLVESGGGLVQAGGSLRLSCAASGRTV SDMTMGWFRQAPGKERVFVAAISNSGLSTYYQDSVKGRFTISRDTANNTVALQMNSLK PEDTAVYFCAARSGWSGQYDYWGQGTQ VTVSS >108G10RAGE 314 EVQLVESGGGLVQAGGSLRLSCAASGRIF NNYAMGWFRQAPGKEREFVAGISWSGDSTLYADSVKGRFTTSRDNAKNTVYLQMNS LKPEDTANYYCAEKQGADWAPYDYWGQGTQVTVSS >108G12 RAGE 315 EVQLVESGGGLVQAGGSLRLSCVASELTFSLYRMGWFRQAPGKEREFVSAMSTSGAG TYYADSVKGRFTISRDNPKNTVYLQMNSLKPEDTAVYYCVAGVRFGVYDYWGQGTQ VTVSS >98E7 Lox- 316EVQLVESGGGLVQPGGSLRLSCAASGFTL 1 DDYAIGWFRQAPGKEREGVSCISRTDGSTDYADSVKGRFTISRDNAKNTVYLQMNSL KPEDTAVYYCAAGRTYYSGSYYFGLGSDEYDYWGQGTQVTVSS >98D8 Lox- 317 EVQLVESGGGLVQPGGSLRLSCAASGSIFT 1INAMAWYRQAPGKQRELVAHLTNSGRTG YADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCNRLGLHWSWGQGTQVTVSS >98B9 Lox- 318EVQLVESGGGLVQAGGSLRLSCAASIGTFS 1 AYHMGWERQAPGKERELVAAISWSVSSTYYADSVKGRFTISRDNAKRTVSLQMDSLK PEDTAVYYCAARSGERYDYYKAQYEYWGQGTQVTVSS >98E9 Lox- 319 EVQLVESGGGLVQPGGSLRLSCAAYGSFF 1SIGTMGWYRQPPGNQRELVAVTYGLGST NYAESVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCYAEIDTDPRSGEWDYWGQG TQVTVSS >98A10 Lox- 320EVQLVESGGGLVQPGGSLRLSCLPSTSTSS 1 LRTVGWYRQGPGKQRDLVAIMSAGTTRYADSVKGRFTISLDDAKNTVYLQMNSLKPE DTAVYICNGRPVFSNVDYWGQGTQVTVS S >98F12 Lox-321 EVQLVESGGGLVQAGGSLRLSCAASGFTF 1 DDYAIGWFRQAPGKEREGVSCVSRDGGSTYYLDSVKGRFTISSDNAKNTVYLQMNSLK PEDAAVYYCAASRYDCSKYLIDYNYRGQGTQVTVSS >105B5 Lox- 322 EVQLVKSGGGLVQAGGSLRLSCAASGRRF 1STSGMGWFRQAPGREREFV?GI?WNSR?TY YAESVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCATNYYGS?WSVNSDDYDYW? QG?QVTVSS >105F7 Lox- 323EVQLVESGGGLVQAGGSLRLSCAASGRTF 1 SNYAMGWFRQAPGKEREFVAAITWSGSSTYYADSVKGRFTISRDNAKNTVYLQMNSL KPEDTAVYYCAAAQRGRYYYLDRNVEYDYWGQGTQVTVSS >105G7 Lox- 324 EVQLVESGGGLVQPGGSLRLSCAASGFTL 1DDYGIGWFRQAPGKEREGVSCISSSDGST DYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCAAGRTYYSGSYYFGLGSD EYDYWGQGTQVTVSS >105A8 Lox- 325EVQLVESGGNLVQAGGSLRLSCAASGFTF 1 DDYVIGWFRQAPGKEREGVSCISSVEGSTYYADSVKGRFTISGDNAKNTVYLQMNSL KPEDTAVYYCAAGTWLDCSGYGSYDMDYWGKGTLVTVSS >105B8 Lox- 326 EVQLVESGGGLVQAGGSLRLSCAASGFTF 1DDYVIGWFRQAPGKEREGVSCISSSEGSTY YAESVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASTWLDFVHGNEYDYRGQ GTQVTVSS >105A11 SR- 327EVQLVESGGGLVQAGGSLRLSCTASGRAV A1 STYAMGWFRQAPGKEREFVAAMISSLSSKSYADTVKGRFTISRDYAKNTVY?QMNSLK PEDTADYYCAADLLPYSSSRSLPMGYDYWGQGTQVTVSS >105C11 SR- 328 EVQLVESGGGLVQAGGSLRLSCTASGRAV A1STYAMGWFRQAPGKEREFVAAMISSLSSK SYADSVKGRFTISRDYAKNTVYLQMNSLKPEDTADYYCAADLLPYSSTRSLPMGYDY WGQGTQVTVSS >105D9 SR- 329EVQLVESGGGLVQAGGSLRLSCAASGSFS A1 LYDMGWESQAPGKEREEVAAINWSGGSTAYADSVKGRFTISRDSAKNTVYLQMNSLK PEDTAVYYCAAKPAKYHFGSGYRDFAEYPYWGQGTQVTVSS >105F12 SR- 330 EVQLVESGGGLVQAGGSLRLSCAASGRTF A1SRYAMAWFRHAPGKDREFVAAVSQSGLL TFYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYDCAA?SRFPLVVPVAYENWG QGTQVTVSS >105G11 SR- 331EVQLVESGGGLVQAGGSLRLSCAASGRTF A1 SRYAMAWFRHAPGKDREFVAAVSQSGLLTFYADSVKGRFTISRDNAKNTVYLQMNSL KPEDTAVYDCAADSRFPLVVPVAYENWGQGTQVTVSS >99C9 SR- 332 EVQLVESGGGLVQVGGSLRLSCAASGISIR A1THAMGWYRQAPGKQRELVATITSVTSGG SLNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKLLGFDYRGQGTQVTVSS >99C8 SR- 333EVQLVESGGGLVQPGGSLRLSCAASGSIG A1 REVAMGWYRQAPGKQRELVATITSITSGGRTNYADSVKGRFTISRDNAKNTVYLQMN SLKPEDTAVYYCNVVPYVNDYWGQGTQ VTVSSwherein RAGE target is human RAGE (SEQ ID NO: 334—Genbank reference:NM_(—)014226):MKNYKAIGKIGEGTFSEVMKM QSLRDGNYYACKQMKQRFESIEQVNNLREIQALRRLNPHPNILMLHEVVFDRKSGSLALICELMDMNIYELIRGRRYPLSEKKIMHYMYQLCKSLDHIHRNGIFHRDVKPENILIKQDVLKLGDFGSCRSVYSKQPYTEYISTRWYRAPECLLTDGFYTYKMDLWSAGCVFYEIASLQPLFPGVNELDQISKIHDVIGTPAQKILTKFKQSRAMNFDFPFKKGSGIPLLTTNLSPQCLSLLHAMVAYDPDERIAAHQALQHPYFQEQRKTEKRALGSHRKAGFPEHPVAPEPLSNSCQISKEGRKQKQSLKQEEDRPKRRGPAYVMELPKLKLSGVVRLSSYSSPTLQSVLGSGTNGRVPVLRPLKCIPASKKTDPQKDLKPAPQQCRLPTIVRKGGR;wherein Lox-1 target is human Lox-1 (SEQ ID NO: 335—Genbank reference:NM_(—)002543):MTFDDLKIQTVKDQPDEKSNGKKAKGLQFLYSPWWCLAAATLGVLCLGLVVTIMVLGMQLSQVSDLLTQEQANLTHQKKKLEGQISARQQAEEASQESENELKEMIETLARKLNEKSKEQMELHHQNLNLQETLKRVANCSAPCPQDWIWHGENCYLFSSGSFNWEKSQEKCLSLDAKLLKINSTADLDFIQQAISYSSFPFWMGLSRRNPSYPWLWEDGSPLMPHLFRVRGAVSQTYPSGTCAYIQRGAVYAENCILAAFSICQKKANLRAQwherein SR-A1 target is human SR-A1 (SEQ ID NO: 336—Genbank reference:NM_(—)021228):MEEEDESRGKTEESGEDRGDGPPDRDPTLSPSAFILRAIQQAVGSSLQGDLPNDKDGSRCHGLRWRRCRSPRSEPRSQESGGTDTATVLDMATDSFLAGLVSVLDPPDTWVPSRLDLRPGESEDMLELVAEVRIGDRDPIPLPVPSLLPRLRAWRTGKTVSPQSNSSRPTCARHLTLGTGDGGPAPPPAPSSASSSPSPSPSSSSPSPPPPPPPPAPPAPPAPRFDIYDPFHPTDEAYSPPPAPEQKYDPFEPTGSNPSSSAGTPSPEEEEEEEEEEEEEEEDEEEEEGLSQSISRISETLAGIYDDNSLSQDFPGDESPRPDAQPTQPTPAPGTPPQVDSTRADGAMRRRVFVVGTEAEACREGKVSVEVVTAGGAALPPPLLPPGDSEIEEGEIVQPEEEPRLALSLFRPGGRAARPPPAASATPTAQPLPQPPAPRAPEGDDELSLHAESDGEGALQVDLGEPAPAPPAADSRWGGLDLRRKILTQRRERYRQRSPSPAPAPAPAAAAGPPTRKKSRRERKRSGEAKEAASSSSGTQPAPPAPASPWDSKKHRSRDRKPGSHASSSARRRSRSRSRSRSTRRRSRSTDRRRGGSRRSRSREKRRRRRRSASPPPATSSSSSSRRERHRGKHRDGGGSKKKKKRSRSRGEKRSGDGSEKAPAPAPPPSGSTSCGDRDSRRRGAVPPSIQDLTDHDLFAIKRTITVGRLDKSDPRGPSPAPASSPKREVLYDSEGLSGEERGGKSSQKDRRRSGAASSSSSSREKGSRRKALDGGDRDRDRDRDRDRDRSSKKARPPKESAPSSGPPPKPPVSSGSGSSSSSSSCSSRKVKLQSKVAVLIREGVSSTTPAKDAASAGLGSIGVKFSRDRESRSPFLKPDERAPTEMAKAAPGSTKPKKTKVKAKAGAKKTKGTKGKTKPSKTRKKVRSGGGSGGSGGQVSLKKSKADSCSQAAGTKGAEETSWSGEERAAKVPSTPPPKAAPPPPALTPDSQTVDSSCKTPEVSFLPEEATEEAGVRGGAEEEEEEEEEEEEEEEEEEQQPATTTATSTAAAAPSTAPSAGSTAGDSGAEDGPASRVSQLPTLPPPMPWNLPAGVDCTTSGVLALTALLFKMEEANLASRAKAQELIQATNQILSHRKPPSSLGMTPAPVPTSLGLPPGPSSYLLPGSLPLGGCGSTPPTPTGLAATSDKREGSSSSEGRGDTDKYLKKLHTQERAVEEVKLAIKPYYQKKDITKEEYKDILRKAVHKICHSKSGEINPVKVSNLVRAYVQRYRYFRKHGRKPGDPPGPPRPPKEPGPPDKGGPGLPLPPL

In particular, the invention in some specific aspects provides:

-   -   amino acid sequences that are directed against (as defined        herein) multiscavenger receptors and that have at least 80%,        preferably at least 85%, such as 90% or 95% or more sequence        identity with at least one of the amino acid sequences of SEQ ID        NO's: 308-333 (see Table A-1). These amino acid sequences may        further be such that they neutralize binding of the cognate        ligand to multiscavenger receptors; and/or compete with the        cognate ligand for binding to multiscavenger receptors; and/or        are directed against an interaction site (as defined herein) on        multiscavenger receptors (such as the ligand binding site);    -   amino acid sequences that cross-block (as defined herein) the        binding of at least one of the amino acid sequences of SEQ ID        NO's: 308-333 (see Table A-1) to multiscavenger receptors and/or        that compete with at least one of the amino acid sequences of        SEQ ID NO's: 308-333 (see Table A-1) for binding to        multiscavenger receptors. Again, these amino acid sequences may        further be such that they neutralize binding of the cognate        ligand to multiscavenger receptors; and/or compete with the        cognate ligand for binding to multiscavenger receptors; and/or        are directed against an interaction site (as defined herein) on        multiscavenger receptors (such as the ligand binding site);        which amino acid sequences may be as further described herein        (and may for example be Nanobodies); as well as polypeptides of        the invention that comprise one or more of such amino acid        sequences (which may be as further described herein, and may for        example be bispecific and/or biparatopic polypeptides as        described herein), and nucleic acid sequences that encode such        amino acid sequences and polypeptides. Such amino acid sequences        and polypeptides do not include any naturally occurring ligands.

In some other specific aspects, the invention provides:

-   -   amino acid sequences of the invention that are specific for        multiscavenger receptors compared to CD36, SR-B1, and galectin-3        and in particular their human sequences;        which amino acid sequences of the invention may be as further        described herein (and may for example be Nanobodies); as well as        polypeptides of the invention that comprise one or more of such        amino acid sequences (which may be as further described herein,        and may for example be bispecific and/or biparatopic        polypeptides as described herein), and nucleic acid sequences        that encode such amino acid sequences and polypeptides. Such        amino acid sequences and polypeptides do not include any        naturally occurring ligands.

Accordingly, some particularly preferred Nanobodies of the invention areNanobodies which can bind (as further defined herein) to and/or aredirected against to multiscavenger receptors and which:

-   i) have at least 80% amino acid identity with at least one of the    amino acid sequences of SEQ ID NO's: 308-333 (see Table A-1), in    which for the purposes of determining the degree of amino acid    identity, the amino acid residues that form the CDR sequences are    disregarded. In this respect, reference is also made to Table B-1,    which lists the framework 1 sequences (SEQ ID NO's: 126-151),    framework 2 sequences (SEQ ID NO's: 178-203), framework 3 sequences    (SEQ ID NO's: 230-255) and framework 4 sequences (SEQ ID NO's:    282-307) of the Nanobodies of SEQ ID NO's: 308-333 (see Table A-1)    (with respect to the amino acid residues at positions 1 to 4 and 27    to 30 of the framework 1 sequences, reference is also made to the    comments made below. Thus, for determining the degree of amino acid    identity, these residues are preferably disregarded);    and in which:-   ii) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    B-2 below.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Again, such Nanobodies may be derived in any suitable manner and fromany suitable source, and may for example be naturally occurring V_(HH)sequences (i.e. from a suitable species of Camelid) or synthetic orsemi-synthetic amino acid sequences, including but not limited to“humanized” (as defined herein) Nanobodies, “camelized” (as definedherein) immunoglobulin sequences (and in particular camelized heavychain variable domain sequences), as well as Nanobodies that have beenobtained by techniques such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, veneering, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing as further described herein. Also, when a Nanobodycomprises a V_(HH) sequence, said Nanobody may be suitably humanized, asfurther described herein, so as to provide one or more further(partially or fully) humanized Nanobodies of the invention. Similarly,when a Nanobody comprises a synthetic or semi-synthetic sequence (suchas a partially humanized sequence), said Nanobody may optionally befurther suitably humanized, again as described herein, again so as toprovide one or more further (partially or fully) humanized Nanobodies ofthe invention.

In particular, humanized Nanobodies may be amino acid sequences that areas generally defined for Nanobodies in the previous paragraphs, but inwhich at least one amino acid residue is present (and in particular, inat least one of the framework residues) that is and/or that correspondsto a humanizing substitution (as defined herein). Some preferred, butnon-limiting humanizing substitutions (and suitable combinations thereonwill become clear to the skilled person based on the disclosure herein.In addition, or alternatively, other potentially useful humanizingsubstitutions can be ascertained by comparing the sequence of theframework regions of a naturally occurring V_(HH) sequence with thecorresponding framework sequence of one or more closely related humanV_(H) sequences, after which one or more of the potentially usefulhumanizing substitutions (or combinations thereof) thus determined canbe introduced into said V_(HH) sequence (in any mariner known per se, asfurther described herein) and the resulting humanized V_(HH) sequencescan be tested for affinity for the target, for stability, for ease andlevel of expression, and/or for other desired properties. In this way,by means of a limited degree of trial and error, other suitablehumanizing substitutions (or suitable combinations thereof) can bedetermined by the skilled person based on the disclosure herein. Also,based on the foregoing, (the framework regions of) a Nanobody may bepartially humanized or fully humanized.

Thus, some other preferred Nanobodies of the invention are Nanobodieswhich can bind (as further defined herein) to multiscavenger receptorsand which:

-   i) are a humanized variant of one of the amino acid sequences of SEQ    ID NO's: 308-333 (see Table A-1); and/or-   ii) have at least 80% amino acid identity with at least one of the    amino acid sequences of SEQ ID NO's: 308-333 (see Table A-1) in    which for the purposes of determining the degree of amino acid    identity, the amino acid residues that form the CDR sequences are    disregarded;    and in which:-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    B-2 below.

According to another specific aspect of the invention, the inventionprovides a number of stretches of amino acid residues (i.e. smallpeptides) that are particularly suited for binding to multiscavengerreceptors. These stretches of amino acid residues may be present in,and/or may be corporated into, an amino acid sequence of the invention,in particular in such a way that they form (part of) the antigen bindingsite of an amino acid sequence of the invention. As these stretches ofamino acid residues were first generated as CDR sequences of heavy chainantibodies or V_(HS) sequences that were raised against multiscavengerreceptors (or may be based on and/or derived from such CDR sequences, asfurther described herein), they will also generally be referred toherein as “CDR sequences” (i.e. as CDR1 sequences, CDR2 sequences andCDR3 sequences, respectively). It should however be noted that theinvention in its broadest sense is not limited to a specific structuralrole or function that these stretches of amino acid residues may have inan amino acid sequence of the invention, as long as these stretches ofamino acid residues allow the amino acid sequence of the invention tobind to multiscavenger receptors. Thus, generally, the invention in itsbroadest sense comprises any amino acid sequence that is capable ofbinding to multiscavenger receptors and that comprises one or more CDRsequences as described herein, and in particular a suitable combinationof two or more such CDR sequences, that are suitably linked to eachother via one or more further amino acid sequences, such that the entireamino acid sequence forms a binding domain and/or binding unit that iscapable of binding to multiscavenger receptors. It should however alsobe noted that the presence of only one such CDR sequence in an aminoacid sequence of the invention may by itself already be sufficient toprovide an amino acid sequence of the invention that is capable ofbinding to multiscavenger receptors; reference is for example again madeto the so-called “Expedite fragments” described in WO 03/050531.

Thus, in another specific, but non-limiting aspect, the amino acidsequence of the invention may be an amino acid sequence that comprisesat least one amino acid sequence that is chosen from the groupconsisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences thatare described herein (or any suitable combination thereof). Inparticular, an amino acid sequence of the invention may be an amino acidsequence that comprises at least one antigen binding site, wherein saidantigen binding site comprises at least one amino acid sequence that ischosen from the group consisting of the CDR1 sequences, CDR2 sequencesand CDR3 sequences that are described herein (or any suitablecombination thereof).

Generally, in this aspect of the invention, the amino acid sequence ofthe invention may be any amino acid sequence that comprises at least onestretch of amino acid residues, in which said stretch of amino acidresidues has an amino acid sequence that corresponds to the sequence ofat least one of the CDR sequences described herein. Such an amino acidsequence may or may not comprise an immunoglobulin fold. For example,and without limitation, such an amino acid sequence may be a suitablefragment of an immunoglobulin sequence that comprises at least one suchCDR sequence, but that is not large enough to form a (complete)immunoglobulin fold (reference is for example again made to the“Expedite fragments” described in WO 03/050531). Alternatively, such anamino acid sequence may be a suitable “protein scaffold” that comprisesleast one stretch of amino acid residues that corresponds to such a CDRsequence (i.e. as part of its antigen binding site). Suitable scaffoldsfor presenting amino acid sequences will be clear to the skilled person,and for example comprise, without limitation, to binding scaffolds basedon or derived from immunoglobulins (i.e. other than the immunoglobulinsequences already described herein), protein scaffolds derived fromprotein A domains (such as Affibodies™), tendamistat, fibronectin,lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimersand PDZ domains (Binz et al., Nat. Biotech 2005, Vol 23:1257), andbinding moieties based on DNA or RNA including but not limited to DNA orRNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 20069(8):619-32).

Again, any amino acid sequence of the invention that comprises one ormore of these CDR sequences is preferably such that it can specificallybind (as defined herein) to multiscavenger receptors, and more inparticular such that it can bind to multiscavenger receptors with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein), that is as defined herein.

More in particular, the amino acid sequences according to this aspect ofthe invention may be any amino acid sequence that comprises at least oneantigen binding site, wherein said antigen binding site comprises atleast two amino acid sequences that are chosen from the group consistingof the CDR1 sequences described herein, the CDR2 sequences describedherein and the CDR3 sequences described herein, such that (i) when thefirst amino acid sequence is chosen from the CDR1 sequences describedherein, the second amino acid sequence is chosen from the CDR2 sequencesdescribed herein or the CDR3 sequences described herein; (ii) when thefirst amino acid sequence is chosen from the CDR2 sequences describedherein, the second amino acid sequence is chosen from the CDR1 sequencesdescribed herein or the CDR3 sequences described herein; or (iii) whenthe first amino acid sequence is chosen from the CDR3 sequencesdescribed herein, the second amino acid sequence is chosen from the CDR1sequences described herein or the CDR3 sequences described herein.

Even more in particular, the amino acid sequences of the invention maybe amino acid sequences that comprise at least one antigen binding site,wherein said antigen binding site comprises at least three amino acidsequences that are chosen from the group consisting of the CDR1sequences described herein, the CDR2 sequences described herein and theCDR3 sequences described herein, such that the first amino acid sequenceis chosen from the CDR1 sequences described herein, the second aminoacid sequence is chosen from the CDR2 sequences described herein, andthe third amino acid sequence is chosen from the CDR3 sequencesdescribed herein. Preferred combinations of CDR1, CDR2 and CDR3sequences will become clear from the further description herein. As willbe clear to the skilled person, such an amino acid sequence ispreferably an immunoglobulin sequence (as further described herein), butit may for example also be any other amino acid sequence that comprisesa suitable scaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates toan amino acid sequence directed against multiscavenger receptors, thatcomprises one or more stretches of amino acid residues chosen from thegroup consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 152-177;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;-   d) the amino acid sequences of SEQ ID NO's: 204-229;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;-   g) the amino acid sequences of SEQ ID NO's: 256-281;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of

-   i) the amino acid sequences of SEQ ID NO's: 152-177;-   ii) the amino acid sequences of SEQ ID NO's: 204-229; and-   iii) the amino acid sequences of SEQ ID NO's: 256-281;    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against multiscavenger receptors.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against multiscavenger receptors,that comprises two or more stretches of amino acid residues chosen fromthe group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 152-177;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;-   d) the amino acid sequences of SEQ ID NO's: 204-229;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;-   g) the amino acid sequences of SEQ ID NO's: 256-281;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), f), g), h) or    i); (ii) when the first stretch of amino acid residues corresponds    to one of the amino acid sequences according to d), e) or f), the    second stretch of amino acid residues corresponds to one of the    amino acid sequences according to a), b), c), g), h) or i); or (iii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to g), h) or i), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), d), e) or 1).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the amino acid sequences of SEQ ID NO's: 152-177;-   ii) the amino acid sequences of SEQ ID NO's: 204-229; and-   iii) the amino acid sequences of SEQ ID NO's: 256-281;    such that, (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's:    152-177, the second stretch of amino acid residues corresponds to    one of the amino acid sequences of SEQ ID NO's: 204-229 or of SEQ ID    NO's: 256-281; (ii) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's:    204-229, the second stretch of amino acid residues corresponds to    one of the amino acid sequences of SEQ ID NO's: 152-177 or of SEQ ID    NO's: 256-281; or (iii) when the first stretch of amino acid    residues corresponds to one of the amino acid sequences of SEQ ID    NO's: 256-281, the second stretch of amino acid residues corresponds    to one of the amino acid sequences of SEQ ID NO's: 152-177 or of SEQ    ID NO's: 204-229.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against multiscavenger receptors.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against multiscavenger receptors,that comprises three or more stretches of amino acid residues, in whichthe first stretch of amino acid residues is chosen from the groupconsisting of:

-   a) the amino acid sequences of SEQ ID NO's: 152-177;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the amino acid sequences of SEQ ID NO's: 204-229;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 256-281;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    256-281.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 152-177; the second stretch of amino acid residues ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 204-229; and the third stretch of amino acid residues is chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's:256-281.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against multiscavenger receptors.

Preferred combinations of such stretches of amino acid sequences willbecome clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 308-333 (see Table A-1). This degree of amino acid identity canfor example be determined by determining the degree of amino acididentity (in a manner described herein) between said amino acid sequenceand one or more of the sequences of SEQ ID NO's: 308-333 (see TableA-1), in which the amino acid residues that form the framework regionsare disregarded. Also, such amino acid sequences of the invention can beas further described herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to multiscavenger receptors; andmore in particular bind to multiscavenger receptors with an affinity(suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 152-177;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 204-229;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 256-281;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    256-281.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 152-177; and/or CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 204-229; and/orCDR3 is chosen from the group consisting of the amino acid sequences ofSEQ ID NO's: 256-281.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 152-177;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 204-229;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 256-281;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    256-281; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 152-177; and CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 204-229; and CDR3is chosen from the group consisting of the amino acid sequences of SEQID NO's: 256-281.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to multiscavenger receptors; andmore in particular bind to multiscavenger receptors with an affinity(suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 308-333 (see Table A-1). Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and one or more of thesequences of SEQ ID NO's: 308-333 (see Table A-1), in which the aminoacid residues that form the framework regions are disregarded. Suchamino acid sequences of the invention can be as further describedherein.

In such an amino acid sequence of the invention, the framework sequencesmay be any suitable framework sequences, and examples of suitableframework sequences will be clear to the skilled person, for example onthe basis the standard handbooks and the further disclosure and priorart mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, byhumanization or camelization). For example, the framework sequences maybe framework sequences derived from a light chain variable domain (e.g.a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. aV_(H)-sequence). In one particularly preferred aspect, the frameworksequences are either framework sequences that have been derived from aV_(HH)-sequence (in which said framework sequences may optionally havebeen partially or fully humanzed) or are conventional V_(H) sequencesthat have been camelized (as defined herein).

The framework sequences are preferably such that the amino acid sequenceof the invention is a domain antibody (or an amino acid sequence that issuitable for use as a domain antibody); is a single domain antibody (oran amino acid sequence that is suitable for use as a single domainantibody); is a “dAb” (or an amino acid sequence that is suitable foruse as a dAb); or is a Nanobody (including but not limited to V_(HH)sequence). Again, suitable framework sequences will be clear to theskilled person, for example on the basis the standard handbooks and thefurther disclosure and prior art mentioned herein.

In particular, the framework sequences present in the amino acidsequences of the invention may contain one or more of Hallmark residues(as defined herein), such that the amino acid sequence of the inventionis a Nanobody. Some preferred, but non-limiting examples of (suitablecombinations of) such framework sequences will become clear from thefurther disclosure herein.

Again, as generally described herein for the amino acid sequences of theinvention, it is also possible to use suitable fragments (orcombinations of fragments) of any of the foregoing, such as fragmentsthat contain one or more CDR sequences, suitably flanked by and/orlinked via one or more framework sequences (for example, in the sameorder as these CDR's and framework sequences may occur in the full-sizedimmunoglobulin sequence from which the fragment has been derived). Suchfragments may also again be such that they comprise or can form animmunoglobulin fold, or alternatively be such that they do not compriseor cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequenceas described herein (and in particular a CDR3 sequence), that is flankedon each side by (part of) a framework sequence (and in particular, partof the framework sequence(s) that, in the immunoglobulin sequence fromwhich the fragment is derived, are adjacent to said CDR sequence. Forexample, a CDR3 sequence may be preceded by (part of) a FR3 sequence andfollowed by (part of) a FR4 sequence). Such a fragment may also containa disulphide bridge, and in particular a disulphide bridge that linksthe two framework regions that precede and follow the CDR sequence,respectively (for the purpose of forming such a disulphide bridge,cysteine residues that naturally occur in said framework regions may beused, or alternatively cysteine residues may be synthetically added toor introduced into said framework regions). For a further description ofthese “Expedite fragments”, reference is again made to WO 03/050531, aswell as to the US provisional application of Ablynx N.V. entitled“Peptides capable of binding to serum proteins” of Ablynx N.V.(inventors: Revets, Hilde Adi Pierrette; Kolkman, Joost Alexander; andHoogenboom, Hendricus Renerus Jacobus Mattheus) filed on Dec. 5, 2006(see also PCT/EP2007/063348).

In another aspect, the invention relates to a compound or construct, andin particular a protein or polypeptide (also referred to herein as a“compound of the invention” or “polypeptide of the invention”,respectively) that comprises or essentially consists of one or moreamino acid sequences of the invention (or suitable fragments thereof),and optionally further comprises one or more other groups, residues,moieties or binding units. As will become clear to the skilled personfrom the further disclosure herein, such further groups, residues,moieties, binding units or amino acid sequences may or may not providefurther functionality to the amino acid sequence of the invention(and/or to the compound or construct in which it is present) and may ormay not modify the properties of the amino acid sequence of theinvention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional amino acid sequences, such that thecompound or construct is a (fusion) protein or (fusion) polypeptide. Ina preferred but non-limiting aspect, said one or more other groups,residues, moieties or binding units are immunoglobulin sequences. Evenmore preferably, said one or more other groups, residues, moieties orbinding units are chosen from the group consisting of domain antibodies,amino acid sequences that are suitable for use as a domain antibody,single domain antibodies, amino acid sequences that are suitable for useas a single domain antibody, “dAb”'s, amino acid sequences that aresuitable for use as a dAb, or Nanobodies.

Alternatively, such groups, residues, moieties or binding units may forexample be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moreamino acid sequences of the invention so as to provide a “derivative” ofan amino acid sequence or polypeptide of the invention, as furtherdescribed herein.

Also within the scope of the present invention are compounds orconstructs, that comprises or essentially consists of one or morederivatives as described herein, and optionally further comprises one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more aminoacid sequences of the invention and the one or more groups, residues,moieties or binding units may be linked directly to each other and/orvia one or more suitable linkers or spacers. For example, when the oneor more groups, residues, moieties or binding units are amino acidsequences, the linkers may also be amino acid sequences, so that theresulting compound or construct is a fusion (protein) or fusion(polypeptide).

As will be clear from the further description above and herein, thismeans that the amino acid sequences of the invention can be used as“building blocks” to form polypeptides of the invention, i.e. bysuitably combining them with other groups, residues, moieties or bindingunits, in order to form compounds or constructs as described herein(such as, without limitations, the biparatopic. bi/multivalent andbi/multispecific polypeptides of the invention described herein) whichcombine within one molecule one or more desired properties or biologicalfunctions.

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least one step of suitably linking theone or more amino acid sequences of the invention to the one or morefurther groups, residues, moieties or binding units, optionally via theone or more suitable linkers, so as to provide the compound orpolypeptide of the invention. Polypeptides of the invention can also beprepared by a method which generally comprises at least the steps ofproviding a nucleic acid that encodes a polypeptide of the invention,expressing said nucleic acid in a suitable manner, and recovering theexpressed polypeptide of the invention. Such methods can be performed ina manner known per se, which will be clear to the skilled person, forexample on the basis of the methods and techniques further describedherein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be “in the format of” said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedamino acid sequences form a further aspect of the invention.

In one specific aspect of the invention, a compound of the invention ora polypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such compounds and polypeptideswill become clear to the skilled person based on the further disclosureherein, and for example comprise amino acid sequences or polypeptides ofthe invention that have been chemically modified to increase thehalf-life thereof (for example, by means of pegylation); amino acidsequences of the invention that comprise at least one additional bindingsite for binding to a serum protein (such as serum albumin); orpolypeptides of the invention that comprise at least one amino acidsequence of the invention that is linked to at least one moiety (and inparticular at least one amino acid sequence) that increases thehalf-life of the amino acid sequence of the invention. Examples ofpolypeptides of the invention that comprise such half-life extendingmoieties or amino acid sequences will become clear to the skilled personbased on the further disclosure herein; and for example include, withoutlimitation, polypeptides in which the one or more amino acid sequencesof the invention are suitable linked to one or more serum proteins orfragments thereof (such as (human) serum albumin or suitable fragmentsthereof) or to one or more binding units that can bind to serum proteins(such as, for example, domain antibodies, amino acid sequences that aresuitable for use as a domain antibody, single domain antibodies, aminoacid sequences that are suitable for use as a single domain antibody,“dAb”'s, amino acid sequences that are suitable for use as a dAb, orNanobodies that can bind to serum proteins such as serum albumin (suchas human serum albumin), serum immunoglobulins such as IgG, ortransferrine; reference is made to the further description andreferences mentioned herein); polypeptides in which an amino acidsequence of the invention is linked to an Fc portion (such as a humanFc) or a suitable part or fragment thereof; or polypeptides in which theone or more amino acid sequences of the invention are suitable linked toone or more small proteins or peptides that can bind to serum proteins(such as, without limitation, the proteins and peptides described in WO91/01743, WO 01/45746, WO 02/076489 and to the US provisionalapplication of Ablynx N.V. entitled “Peptides capable of binding toserum proteins” of Ablynx N.V. filed on. Dec. 5, 2006 (see alsoPCT/EP2007/063348).

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence of the invention per se. For example,the compounds or polypeptides of the invention with increased half-lifemay have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds or polypeptides of the invention exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, compounds or polypeptides of the invention may have ahalf-life of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 1.8 days or more), or more than 14 days (such asabout 14 to 19 days).

In another aspect, the invention relates to a nucleic acid that encodesan amino acid sequence of the invention or a polypeptide of theinvention (or a suitable fragment thereof). Such a nucleic acid willalso be referred to herein as a “nucleic acid of the invention” and mayfor example be in the form of a genetic construct, as further describedherein.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) an amino acid sequence of the invention and/or a polypeptideof the invention; and/or that contains a nucleic acid of the invention.Some preferred but non-limiting examples of such hosts or host cellswill become clear from the further description herein.

The invention further relates to a product or composition containing orcomprising at least one amino acid sequence of the invention, at leastone polypeptide of the invention (or a suitable fragment thereof) and/orat least one nucleic acid of the invention, and optionally one or morefurther components of such compositions known per se, i.e. depending onthe intended use of the composition. Such a product or composition mayfor example be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention, or of a composition comprisingthe same, in (methods or compositions for) modulating multiscavengerreceptors, either in vitro (e.g. in an in vitro or cellular assay) or invivo (e.g. in an a single cell or in a multicellular organism, and inparticular in a mammal, and more in particular in a human being, such asin a human being that is at risk of or suffers from a disease whereinmultiscanvenger receptors are implicated.

The invention also relates to methods for modulating multiscavengerreceptors, either in vitro (e.g. in an in vitro or cellular assay) or invivo (e.g. in an a single cell or multicellular organism, and inparticular in a mammal, and more in particular in a human being, such asin a human being that is at risk of or suffers from a disease whereinmultiscanvenger receptors are implicated), which method comprises atleast the step of contacting multiscavenger receptors with at least oneamino acid sequence, Nanobody or polypeptide of the invention, or with acomposition comprising the same, in a manner and in an amount suitableto modulate multiscavenger receptors, with at least one amino acidsequence, Nanobody or polypeptide of the invention.

The invention also relates to the use of an one amino acid sequence,Nanobody or polypeptide of the invention in the preparation of acomposition (such as, without limitation, a pharmaceutical compositionor preparation as further described herein) for modulatingmultiscavenger receptors, either in vitro (e.g. in an in vitro orcellular assay) or in vivo (e.g. in an a single cell or multicellularorganism, and in particular in a mammal, and more in particular in ahuman being, such as in a human being that is at risk of or suffers froma disease wherein multiscanvenger receptors are implicated).

In the context of the present invention, “modulating” or “to modulate”generally means either reducing or inhibiting the activity of, oralternatively increasing the activity of, multiscavenger receptors, asmeasured using a suitable in vitro, cellular or in vivo assay (such asthose mentioned herein). In particular, “modulating” or “to modulate”may mean either reducing or inhibiting the activity of, or alternativelyincreasing the activity of multiscavenger receptors, as measured using asuitable in vitro, cellular or in vivo assay (such as those mentionedherein), by at least 1%, preferably at least 5%, such as at least 10% orat least 25%, for example by at least 50%, at least 60%, at least 70%,at least 80%, or 90% or more, compared to activity of multiscavengerreceptors in the same assay under the same conditions but without thepresence of the amino acid sequence, Nanobody or polypeptide of theinvention.

As will be clear to the skilled person, “modulating” may also involveeffecting a change (which may either be an increase or a decrease) inaffinity, avidity, specificity and/or selectivity of multiscavengerreceptors for one or more of its targets, ligands or substrates; and/oreffecting a change (which may either be an increase or a decrease) inthe sensitivity of multiscavenger receptors for one or more conditionsin the medium or surroundings in which multiscavenger receptors ispresent (such as pH, ion strength, the presence of co-factors, etc.),compared to the same conditions but without the presence of the aminoacid sequence, Nanobody or polypeptide of the invention. As will beclear to the skilled person, this may again be determined in anysuitable manner and/or using any suitable assay known per se, such asthe assays described herein or in the prior art cited herein.

“Modulating” may also mean effecting a change (i.e. an activity as anagonist or as an antagonist, respectively) with respect to one or morebiological or physiological mechanisms, effects, responses, functions,pathways or activities in which multiscavenger receptors (or in whichits substrate(s), ligand(s) or pathway(s) are involved, such as itssignalling pathway or metabolic pathway and their associated biologicalor physiological effects) is involved. Again, as will be clear to theskilled person, such an action as an agonist or an antagonist may bedetermined in any suitable manner and/or using any suitable (in vitroand usually cellular or in assay) assay known per se, such as the assaysdescribed herein or in the prior art cited herein. In particular, anaction as an agonist or antagonist may be such that an intendedbiological or physiological activity is increased or decreased,respectively, by at least 1%, preferably at least 5%, such as at least10% or at least 25%, for example by at least 50%, at least 60%, at least70%, at least 80%, or 90% or more, compared to the biological orphysiological activity in the same assay under the same conditions butwithout the presence of the amino acid sequence, Nanobody or polypeptideof the invention.

Modulating may for example involve reducing or inhibiting the binding ofmultiscavenger receptors to one of its substrates or ligands and/orcompeting with a natural ligand, substrate for binding to multiscavengerreceptors. Modulating may also involve activating multiscavengerreceptors or the mechanism or pathway in which it is involved.Modulating may be reversible or irreversible, but for pharmaceutical andpharmacological purposes will usually be in a reversible manner.

The invention further relates to methods for preparing or generating theamino acid sequences, polypeptides, nucleic acids, host cells, productsand compositions described herein. Some preferred but non-limitingexamples of such methods will become clear from the further descriptionherein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of amino acid sequences;    and-   b) screening said set, collection or library of amino acid sequences    for amino acid sequences that can bind to and/or have affinity for    multiscavenger receptors;    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for multiscavenger receptors.

In such a method, the set, collection or library of amino acid sequencesmay be any suitable set, collection or library of amino acid sequences.For example, the set, collection or library of amino acid sequences maybe a set, collection or library of immunoglobulin sequences (asdescribed herein), such as a naïve set, collection or library ofimmunoglobulin sequences; a synthetic or semi-synthetic set, collectionor library of immunoglobulin sequences; and/or a set, collection orlibrary of immunoglobulin sequences that have been subjected to affinitymaturation.

Also, in such a method, the set, collection or library of amino acidsequences may be a set, collection or library of heavy chain variabledomains (such as V_(H) domains or V_(HH) domains) or of light chainvariable domains. For example, the set, collection or library of aminoacid sequences may be a set, collection or library of domain antibodiesor single domain antibodies, or may be a set, collection or library ofamino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofimmunoglobulin sequences, for example derived from a mammal that hasbeen suitably immunized with multiscavenger receptors or with a suitableantigenic determinant based thereon or derived therefrom, such as anantigenic part, fragment, region, domain, loop or other epitope thereof.In one particular aspect, said antigenic determinant may be anextracellular part, region, domain, loop or other extracellularepitope(s).

In the above methods, the set, collection or library of amino acidsequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) amino acid sequences will beclear to the person skilled in the art, for example on the basis of thefurther disclosure herein. Reference is also made to the review byHoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating amino acid sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells expressing amino acid    sequences;-   b) screening said collection or sample of cells for cells that    express an amino acid sequence that can bind to and/or have affinity    for multiscavenger receptors;    and-   c) either (i) isolating said amino acid sequence; or (ii) isolating    from said cell a nucleic acid sequence that encodes said amino acid    sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulinsequence, the collection or sample of cells may for example be acollection or sample of B-cells. Also, in this method, the sample ofcells may be derived from a mammal that has been suitably immunized withmultiscavenger receptors or with a suitable antigenic determinant basedthereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820 (2001).

In another aspect, the method for generating an amino acid sequencedirected against multiscavenger receptors may comprise at least thesteps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for multiscavenger    receptors;    and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with multiscavenger receptors or with a suitableantigenic determinant based thereon or derived therefrom, such as anantigenic part, fragment, region, domain, loop or other epitope thereof.In one particular aspect, said antigenic determinant may be anextracellular part, region, domain, loop or other extracellularepitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating an amino acid sequencedirected against multiscavenger receptors may comprise at least thesteps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for multiscavenger    receptors and that is cross-blocked or is cross blocking a Nanobody    of the invention, e.g. SEQ ID NO: 308-333 (Table A-1); and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

The invention also relates to amino acid sequences that are obtained bythe above methods, or alternatively by a method that comprises the oneof the above methods and in addition at least the steps of determiningthe nucleotide sequence or amino acid sequence of said immunoglobulinsequence; and of expressing or synthesizing said amino acid sequence ina manner known per se, such as by expression in a suitable host cell orhost organism or by chemical synthesis.

Also, following the steps above, one or more amino acid sequences of theinvention may be suitably humanized (or alternatively camelized); and/orthe amino acid sequence(s) thus obtained may be linked to each other orto one or more other suitable amino acid sequences (optionally via oneor more suitable linkers) so as to provide a polypeptide of theinvention. Also, a nucleic acid sequence encoding an amino acid sequenceof the invention may be suitably humanized (or alternatively camelized)and suitably expressed; and/or one or more nucleic acid sequencesencoding an amino acid sequence of the invention may be linked to eachother or to one or more nucleic acid sequences that encode othersuitable amino acid sequences (optionally via nucleotide sequences thatencode one or more suitable linkers), after which the nucleotidesequence thus obtained may be suitably expressed so as to provide apolypeptide of the invention.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with multiscavenger receptors. Some preferred butnon-limiting applications and uses will become clear from the furtherdescription herein.

The invention also relates to the amino acid sequences, compounds,constructs, polypeptides, nucleic acids, host cells, products andcompositions described herein for use in therapy.

In particular, the invention also relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of a disease ordisorder that can be prevented or treated by administering, to a subjectin need thereof, of (a pharmaceutically effective amount of) an aminoacid sequence, compound, construct or polypeptide as described herein.

More in particular, the invention relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of a diseasewherein multiscanvenger receptors are implicated.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description herein, in which theinvention will be described and discussed in more detail with referenceto the Nanobodies of the invention and polypeptides of the inventioncomprising the same, which form some of the preferred aspects of theinvention.

As will become clear from the further description herein, Nanobodiesgenerally offer certain advantages (outlined herein) compared to “dAb's”or similar (single) domain antibodies or immunoglobulin sequences, whichadvantages are also provided by the Nanobodies of the invention.However, it will be clear to the skilled person that the more generalaspects of the teaching below can also be applied (either directly oranalogously) to other amino acid sequences of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the present description, examples and claims:

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks mentioned in    paragraph a) on page 46 of WO 08/020,079.-   b) Unless indicated otherwise, the terms “immunoglobulin sequence”,    “sequence”, “nucleotide sequence” and “nucleic acid” are as    described in paragraph b) on page 46 of WO 08/020,079.-   c) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta, Adv. Drug Deliv. Rev.    2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):    49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;    Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et    al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for    protein engineering, such as affinity maturation and other    techniques for improving the specificity and other desired    properties of proteins such as immunoglobulins.-   d) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code. Reference is made to    Table A-2 on page 48 of the International application WO 08/020,079    of Ablynx N.V. entitled “Amino acid sequences directed against IL-6R    and polypeptides comprising the same for the treatment of diseases    and disorders associated with Il-6 mediated signalling”.-   e) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated or    determined as described in paragraph e) on page 49 of WO 08/020,079    (incorporated herein by reference), such as by dividing [the number    of nucleotides in the first nucleotide sequence that are identical    to the nucleotides at the corresponding positions in the second    nucleotide sequence] by [the total number of nucleotides in the    first nucleotide sequence] and multiplying by [100%], in which each    deletion, insertion, substitution or addition of a nucleotide in the    second nucleotide sequence—compared to the first nucleotide    sequence—is considered as a difference at a single nucleotide    (position); or using a suitable computer algorithm or technique,    again as described in paragraph e) on pages 49 of WO 08/020,079    (incorporated herein by reference).-   f) For the purposes of comparing two or more amino acid sequences,    the percentage of “sequence identity” between a first amino acid    sequence and a second amino acid sequence (also referred to herein    as “amino acid identity”) may be calculated or determined as    described in paragraph f) on pages 49 and 50 of WO 08/020,079    (incorporated herein by reference), such as by dividing [the number    of amino acid residues in the first amino acid sequence that are    identical to the amino acid residues at the corresponding positions    in the second amino acid sequence] by [the total number of amino    acid residues in the first amino acid sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of an amino acid residue in the second amino acid sequence—compared    to the first amino acid sequence—is considered as a difference at a    single amino acid residue (position), i.e. as an “amino acid    difference” as defined herein; or using a suitable computer    algorithm or technique, again as described in paragraph f) on pages    49 and 50 of WO 08/020,079 (incorporated herein by reference).

Also, in determining the degree of sequence identity between two aminoacid sequences, the skilled person may take into account so-called“conservative” amino acid substitutions, as described on page 50 of WO08/020,079.

Any amino acid substitutions applied to the polypeptides describedherein may also be based on the analysis of the frequencies of aminoacid variations between homologous proteins of different speciesdeveloped by Schulz et al., Principles of Protein Structure,Springer-Verlag, 1978, on the analyses of structure forming potentialsdeveloped by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv.Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicitypatterns in proteins developed by Eisenberg et al., Proc. Nad. Acad.Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J. Molec. Biol. 157:105-132, 198 1, and Goldman et al., Ann. Rev. Biophys. Chem. 15:321-353, 1986, all incorporated herein in their entirety by reference.Information on the primary, secondary and tertiary structure ofNanobodies is given in the description herein and in the generalbackground art cited above. Also, for this purpose, the crystalstructure of a V_(HH) domain from a llama is for example given byDesmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996);Spinelli et at., Natural Structural Biology (1996); 3, 752-757; andDecanniere et al., Structure, Vol. 7, 4, 361 (1999). Further informationabout some of the amino acid residues that in conventional V₁ domainsform the V_(H)/V_(L) interface and potential camelizing substitutions onthese positions can be found in the prior art cited above.

-   g) Amino acid sequences and nucleic acid sequences are said to be    “exactly the same” if they have 100% sequence identity (as defined    herein) over their entire length.-   h) When comparing two amino acid sequences, the term “amino acid    difference” refers to an insertion, deletion or substitution of a    single amino acid residue on a position of the first sequence,    compared to the second sequence; it being understood that two amino    acid sequences can contain one, two or more such amino acid    differences.-   i) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this has the meaning given in    paragraph i) on pages 51-52 of WO 08/020,079.-   j) The term “in essentially isolated form” has the meaning given to    it in paragraph j) on pages 52 and 53 of WO 08/020,079.-   k) The terms “domain” and “binding domain” have the meanings given    to it in paragraph k) on page 53 of WO 08/020,079.-   l) The terms “antigenic determinant” and “epitope”, which may also    be used interchangeably herein, have the meanings given to it in    paragraph 1) on page 53 of WO 08/020,079.-   m) As further described in paragraph m) on page 53 of WO 08/020,079,    an amino acid sequence (such as a Nanobody, an antibody, a    polypeptide of the invention, or generally an antigen binding    protein or polypeptide or a fragment thereof) that can    (specifically) bind to, that has affinity for and/or that has    specificity for a specific antigenic determinant, epitope, antigen    or protein (or for at least one part, fragment or epitope thereof)    is said to be “against” or “directed against” said antigenic    determinant, epitope, antigen or protein.-   n) The term “specificity” has the meaning given to it in    paragraph n) on pages 53-56 of WO 08/020,079; and as mentioned    therein refers to the number of different types of antigens or    antigenic determinants to which a particular antigen-binding    molecule or antigen-binding protein (such as a Nanobody or a    polypeptide of the invention) molecule can bind. The specificity of    an antigen-binding protein can be determined based on affinity    and/or avidity, as described on pages 53-56 of WO 08/020,079    (incorporated herein by reference), which also describes some    preferred techniques for measuring binding between an    antigen-binding molecule (such as a Nanobody or polypeptide of the    invention) and the pertinent antigen. Typically, antigen-binding    proteins (such as the amino acid sequences. Nanobodies and/or    polypeptides of the invention) will bind to their antigen with a    dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less,    and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably    10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association constant (K_(A))    of 10⁵ to 10¹² liter/moles or more, and preferably 10⁷ to 10¹²    liter/moles or more and more preferably 10⁸ to 10¹² liter/moles).    Any K_(D) value greater than 10⁴ mol/liter (or any K_(A) value lower    than 10⁴ M⁻¹) liters/mol is generally considered to indicate    non-specific binding. Preferably, a monovalent immunoglobulin    sequence of the invention will bind to the desired antigen with an    affinity less than 500 nM, preferably less than 200 nM, more    preferably less than 10 nM, such as less than 500 pM. Specific    binding of an antigen-binding protein to an antigen or antigenic    determinant can be determined in any suitable manner known per se,    including, for example, Scatchard analysis and/or competitive    binding assays, such as radioimmunoassays (RIA), enzyme immunoassays    (EIA) and sandwich competition assays, and the different variants    thereof known per se in the art; as well as the other techniques    mentioned herein. As will be clear to the skilled person, and as    described on pages 53-56 of WO 08/020,079, the dissociation constant    may be the actual or apparent dissociation constant. Methods for    determining the dissociation constant will be clear to the skilled    person, and for example include the techniques mentioned on pages    53-56 of WO 08/020,079.-   o) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as described in    paragraph o) on page 57 of WO 08/020,079 and as mentioned therein    refers to the time taken for the serum concentration of the amino    acid sequence, compound or polypeptide to be reduced by 50%, in    vivo, for example due to degradation of the sequence or compound    and/or clearance or sequestration of the sequence or compound by    natural mechanisms. The in vivo half-life of an amino acid sequence,    compound or polypeptide of the invention can be determined in any    manner known per se, such as by pharmacokinetic analysis. Suitable    techniques will be clear to the person skilled in the art, and may    for example generally be as described in paragraph o) on page 57 of    WO 08/020,079. As also mentioned in paragraph o) on page 57 of WO    08/020,079, the half-life can be expressed using parameters such as    the t1/2-alpha, t1/2-beta and the area under the curve (AUC).    Reference is for example made to the Experimental Part below, as    well as to the standard handbooks, such as Kenneth, A et al:    Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists    and Peters et al, Pharmacokinete analysis: A Practical Approach    (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D    Perron, published by Marcel Dekker, 2nd Rev. edition (1982). The    terms “increase in half-life” or “increased half-life” as also as    defined in paragraph o) on page 57 of WO 08/020,079 and in    particular refer to an increase in the t1/2-beta, either with or    without an increase in the t1/2-alpha and/or the AUC or both.-   p) In the context of the present invention, “modulating” or “to    modulate” generally means either reducing or inhibiting the activity    of, or alternatively increasing the activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay. In particular, “modulating” or “to modulate” may mean either    reducing or inhibiting the activity of, or alternatively increasing    a (relevant or intended) biological activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay (which will usually depend on the target or antigen involved),    by at least 1%, preferably at least 5%, such as at least 10% or at    least 25%, for example by at least 50%, at least 60%, at least 70%,    at least 80%, or 90% or more, compared to activity of the target or    antigen in the same assay under the same conditions but without the    presence of the construct of the invention.

As will be clear to the skilled person, “modulating” may also involveeffecting a change (which may either be an increase or a decrease) inaffinity, avidity, specificity and/or selectivity of a target or antigenfor one or more of its ligands, binding partners, partners forassociation into a homomultimeric or heteromultimeric form, orsubstrates; and/or effecting a change (which may either be an increaseor a decrease) in the sensitivity of the target or antigen for one ormore conditions in the medium or surroundings in which the target orantigen is present (such as pH, ion strength, the presence ofco-factors, etc.), compared to the same conditions but without thepresence of the construct of the invention. As will be clear to theskilled person, this may again be determined in any suitable trimmerand/or using any suitable assay known per se, depending on the target orantigen involved.

“Modulating” may also mean effecting a change (i.e. an activity as anagonist, as an antagonist or as a reverse agonist, respectively,depending on the target or antigen and the desired biological orphysiological effect) with respect to one or more biological orphysiological mechanisms, effects, responses, functions, pathways oractivities in which the target or antigen (or in which its substrate(s),ligand(s) or pathway(s) are involved, such as its signalling pathway ormetabolic pathway and their associated biological or physiologicaleffects) is involved. Again, as will be clear to the skilled person,such an action as an agonist or an antagonist may be determined in anysuitable manner and/or using any suitable (in vitro and usually cellularor in assay) assay known per se, depending on the target or antigeninvolved. In particular, an action as an agonist or antagonist may besuch that an intended biological or physiological activity is increasedor decreased, respectively, by at least 1%, preferably at least 5%, suchas at least 10% or at least 25%, for example by at least 50%, at least60%, at least 70%, at least 80%, or 90% or more, compared to thebiological or physiological activity in the same assay under the sameconditions but without the presence of the construct of the invention.

Modulating may for example also involve allosteric modulation of thetarget or antigen; and/or reducing or inhibiting the binding of thetarget or antigen to one of its substrates or ligands and/or competingwith a natural ligand, substrate for binding to the target or antigen.Modulating may also involve activating the target or antigen or themechanism or pathway in which it is involved. Modulating may for examplealso involve effecting a change in respect of the folding orconfirmation of the target or antigen, or in respect of the ability ofthe target or antigen to fold, to change its confirmation (for example,upon binding of a ligand), to associate with other (sub)units, or todisassociate.

Modulating may for example also involve effecting a change in theability of the target or antigen to transport other compounds or toserve as a channel for other compounds (such as ions).

Modulating may be reversible or irreversible, but for pharmaceutical andpharmacological purposes will usually be in a reversible manner.

-   q) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence or polypeptide of the invention can bind such that the    target or antigen (and/or any pathway, interaction, signalling,    biological mechanism or biological effect in which the target or    antigen is involved) is modulated (as defined herein).-   r) An amino acid sequence or polypeptide is said to be “specific    for” a first target or antigen compared to a second target or    antigen when is binds to the first antigen with an affinity (as    described above, and suitably expressed as a K_(D) value, K_(A)    value, K_(off) rate and/or K_(on) rate) that is at least 10 times,    such as at least 100 times, and preferably at least 1000 times, and    up to 10.000 times or more better than the affinity with which said    amino acid sequence or polypeptide binds to the second target or    polypeptide. For example, the first antigen may bind to the target    or antigen with a K_(D) value that is at least 10 times less, such    as at least 100 times less, and preferably at least 1000 times less,    such as 10.000 times less or even less than that, than the K_(D)    with which said amino acid sequence or polypeptide binds to the    second target or polypeptide. Preferably, when an amino acid    sequence or polypeptide is “specific for” a first target or antigen    compared to a second target or antigen, it is directed against (as    defined herein) said first target or antigen, but not directed    against said second target or antigen.-   s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are    used interchangeably herein to mean the ability of an amino acid    sequence or other binding agents (such as a Nanobody, polypeptide or    compound or construct of the invention) to interfere with the    binding of other amino acid sequences or binding agents of the    invention to a given target. The extend to which an amino acid    sequence or other binding agents of the invention is able to    interfere with the binding of another to a given target, and    therefore whether it can be said to cross-block according to the    invention, can be determined using competition binding assays. One    particularly suitable quantitative cross-blocking assay uses a    Biacore machine which can measure the extent of interactions using    surface plasmon resonance technology. Another suitable quantitative    cross-blocking assay uses an ELISA-based approach to measure    competition between amino acid sequences or other binding agents in    terms of their binding to the target.

The following generally describes a suitable Biacore assay fordetermining whether an amino acid sequence or other binding agentcross-blocks or is capable of cross-blocking according to the invention.It will be appreciated that the assay can be used with any of the aminoacid sequences or other binding agents described herein. The Biacoremachine (for example the Biacore 3000) is operated in line with themanufacturer's recommendations. Thus in one cross-blocking assay, thetarget protein is coupled to a CM5 Biacore chip using standard aminecoupling chemistry to generate a surface that is coated with the target.Typically 200-800 resonance units of the target would be coupled to thechip (an amount that gives easily measurable levels of binding but thatis readily saturable by the concentrations of test reagent being used).Two test amino acid sequences (termed A* and B*) to be assessed fortheir ability to cross-block each other are mixed at a one to one molarratio of binding sites in a suitable buffer to create the test mixture.When calculating the concentrations on a binding site basis themolecular weight of an amino acid sequence is assumed to be the totalmolecular weight of the amino acid sequence divided by the number oftarget binding sites on that amino acid sequence. The concentration ofeach amino acid sequence in the test mix should be high enough toreadily saturate the binding sites for that amino acid sequence on thetarget molecules captured on the Biacore chip. The amino acid sequencesin the mixture are at the same molar concentration (on a binding basis)and that concentration would typically be between 1.00 and 1.5micromolar (on a binding site basis). Separate solutions containing A*alone and B* alone are also prepared. A* and B* in these solutionsshould be in the same buffer and at the same concentration as in thetest mix. The test mixture is passed over the target-coated Biacore chipand the total amount of binding recorded. The chip is then treated insuch a way as to remove the bound amino acid sequences without damagingthe chip-bound target. Typically this is done by treating the chip with30 mM HCl for 60 seconds. The solution of A* alone is then passed overthe target-coated surface and the amount of binding recorded. The chipis again treated to remove all of the bound amino acid sequences withoutdamaging the chip-bound target. The solution of B* alone is then passedover the target-coated surface and the amount of binding recorded. Themaximum theoretical binding of the mixture of A* and B* is nextcalculated, and is the sum of the binding of each amino acid sequencewhen passed over the target surface alone. If the actual recordedbinding of the mixture is less than this theoretical maximum then thetwo amino acid sequences are cross-blocking each other. Thus, ingeneral, a cross-blocking amino acid sequence or other binding agentaccording to the invention is one which will bind to the target in theabove Biacore cross-blocking assay such that, during the assay and inthe presence of a second amino acid sequence or other binding agent ofthe invention, the recorded binding is between 80% and 0.1% (e.g. 80% to4%) of the maximum theoretical binding, specifically between 75% and0.1% (e.g. 75% to 4%) of the maximum theoretical binding, and morespecifically between 70% and 0.1% (e.g. 70% to 4%) of maximumtheoretical binding (as just defined above) of the two amino acidsequences or binding agents in combination. The Biacore assay describedabove is a primary assay used to determine if amino acid sequences orother binding agents cross-block each other according to the invention.On rare occasions particular amino acid sequences or other bindingagents may not bind to target coupled via amine chemistry to a CM5Biacore chip (this usually occurs when the relevant binding site ontarget is masked or destroyed by the coupling to the chip). In suchcases cross-blocking can be determined using a tagged version of thetarget, for example a N-terminal His-tagged version. In this particularformat, an anti-His amino acid sequence would be coupled to the Biacorechip and then the His-tagged target would be passed over the surface ofthe chip and captured by the anti-His amino acid sequence. The crossblocking analysis would be carried out essentially as described above,except that after each chip regeneration cycle, new His-tagged targetwould be loaded back onto the anti-His amino acid sequence coatedsurface. In addition to the example given using N-terminal His-taggedtarget, C-terminal His-tagged target could alternatively be used.Furthermore, various other tags and tag binding protein combinationsthat are known in the art could be used for such a cross-blockinganalysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti-FLAGantibodies; biotin tag with streptavidin).

The following generally describes an ELISA assay for determining whetheran amino acid sequence or other binding agent directed against a targetcross-blocks or is capable of cross-blocking as defined herein. It willbe appreciated that the assay can be used with any of the amino acidsequences (or other binding agents such as polypeptides of theinvention) described herein. The general principal of the assay is tohave an amino acid sequence or binding agent that is directed againstthe target coated onto the wells of an ELISA plate. An excess amount ofa second, potentially cross-blocking, anti-target amino acid sequence isadded in solution (i.e. not bound to the ELISA plate). A limited amountof the target is then added to the wells. The coated amino acid sequenceand the amino acid sequence in solution compete for binding of thelimited number of target molecules. The plate is washed to remove excesstarget that has not been bound by the coated amino acid sequence and toalso remove the second, solution phase amino acid sequence as well asany complexes formed between the second, solution phase amino acidsequence and target. The amount of bound target is then measured using areagent that is appropriate to detect the target. An amino acid sequencein solution that is able to cross-block the coated amino acid sequencewill be able to cause a decrease in the number of target molecules thatthe coated amino acid sequence can bind relative to the number of targetmolecules that the coated amino acid sequence can bind in the absence ofthe second, solution phase, amino acid sequence. In the instance wherethe first amino acid sequence, e.g. an Ab-X, is chosen to be theimmobilized amino acid sequence, it is coated onto the wells of theELISA plate, after which the plates are blocked with a suitable blockingsolution to minimize non-specific binding of reagents that aresubsequently added. An excess amount of the second amino acid sequence,i.e. Ab-Y, is then added to the ELISA plate such that the moles of Ab-Ytarget binding sites per well are at least 10 fold higher than the molesof Ab-X target binding sites that were used, per well, during thecoating of the ELISA plate. Target is then added such that the moles oftarget added per well are at least 25-fold lower than the moles of Ab-Xtarget binding sites that were used for coating each well. Following asuitable incubation period the ELISA plate is washed and a reagent fordetecting the target is added to measure the amount of targetspecifically bound by the coated anti[target amino acid sequence (inthis case Ab-X). The background signal for the assay is defined as thesignal obtained in wells with the coated amino acid sequence (in thiscase Ab-X), second solution phase amino acid sequence (in this caseAb-Y), target buffer only (i.e. without target) and target detectionreagents. The positive control signal for the assay is defined as thesignal obtained in wells with the coated amino acid sequence (in thiscase Ab-X), second solution phase amino acid sequence buffer only (i.e.without second solution phase amino acid sequence), target and targetdetection reagents. The ELISA assay may be run in such, a manner so asto have the positive control signal be at least 6 times the backgroundsignal. To avoid any artefacts (e.g. significantly different affinitiesbetween Ab-X and Ab-Y for the target) resulting from the choice of whichamino acid sequence to use as the coating amino acid sequence and whichto use as the second (competitor) amino acid sequence, thecross-blocking assay may to be run in two formats: 1) format 1 is whereAb-X is the amino acid sequence that is coated onto the ELISA plate andAb-Y is the competitor amino acid sequence that is in solution and 2)format 2 is where Ab-Y is the amino acid sequence that is coated ontothe ELISA plate and Ab-X is the competitor amino acid sequence that isin solution. Ab-X and Ab-Y are defined as cross-blocking if, either informat 1 or in format 2, the solution phase anti-target amino acidsequence is able to cause a reduction of between 60% and 100%,specifically between 70% and 100%, and more specifically between 80% and100%, of the target detection signal (i.e. the amount of target bound bythe coated amino acid sequence) as compared to the target detectionsignal obtained in the absence of the solution phase anti-target aminoacid sequence (i.e. the positive control wells).

-   t) An amino acid sequence is said to be “cross-reactive” for two    different antigens or antigenic determinants (such as serum albumin    from two different species of mammal, such as human serum albumin    and cyno serum albumin) if it is specific for (as defined herein)    both these different antigens or antigenic determinants.-   u) By binding that is “essentially independent of the pH” is    generally meant herein that the association constant (K_(A)) of the    amino acid sequence with respect to the serum protein (such as serum    albumin) at the pH value(s) that occur in a cell of an animal or    human body (as further described herein) is at least 5%, such as at    least 10%, preferably at least 25%, more preferably at least 50%,    even more preferably at least 60%, such as even more preferably at    least 70%, such as at least 80% or 90% or more (or even more than    100%, such as more than 110%, more than 120% or even 130% or more,    or even more than 150%, or even more than 200%) of the association    constant (K_(A)) of the amino acid sequence with respect to the same    serum protein at the pH value(s) that occur outside said cell.    Alternatively, by binding that is “essentially independent of the    pH” is generally meant herein that the k_(off) rate (measured by    Biacore) of the amino acid sequence with respect to the serum    protein (such as serum albumin) at the pH value(s) that occur in a    cell of an animal or human body (as e.g. further described herein,    e.g. pH around 5.5, e.g. 5.3 to 5.7) is at least 5%, such as at    least 10%, preferably at least 25%, more preferably at least 50%,    even more preferably at least 60%, such as even more preferably at    least 70%, such as at least 80% or 90% or more (or even more than    100%, such as more than 110%, more than 120% or even 130% or more,    or even more than 150%, or even more than 200%) of the k_(off) rate    of the amino acid sequence with respect to the same serum protein at    the pH value(s) that occur outside said cell, e.g. pH 7.2 to 7.4. By    “the pH value(s) that occur in a cell of an animal or human body” is    meant the pH value(s) that may occur inside a cell, and in    particular inside a cell that is involved in the recycling of the    serum protein. In particular, by “the pH value(s) that occur in a    cell of an animal or human body” is meant the pH value(s) that may    occur inside a (sub)cellular compartment or vesicle that is involved    in recycling of the serum protein (e.g. as a result of pinocytosis,    endocytosis, transcytosis, exocytosis and phagocytosis or a similar    mechanism of uptake or internalization into said cell), such as an    endosome, lysosome or pinosome.-   v) As further described herein, the total number of amino acid    residues in a Nanobody can be in the region of 110-120, is    preferably 112-115, and is most preferably 113. It should however be    noted that parts, fragments, analogs or derivatives (as further    described herein) of a Nanobody are not particularly limited as to    their length and/or size, as long as such parts, fragments, analogs    or derivatives meet the further requirements outlined herein and are    also preferably suitable for the purposes described herein;-   w) As further described in paragraph q) on pages 58 and 59 of WO    08/020,079 (incorporated herein by reference), the amino acid    residues of a Nanobody are numbered according to the general    numbering for V_(H) domains given by Kabat et al. (“Sequence of    proteins of immunological interest”, US Public Health Services, NIH    Bethesda, Md., Publication No. 91), as applied to V_(HH) domains    from Camelids in the article of Riechmann and Muyldermans, J.    Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195 (see for example    FIG. 2 of this publication), and accordingly FR1 of a Nanobody    comprises the amino acid residues at positions 1-30, CDR1 of a    Nanobody comprises the amino acid residues at positions 31-35, FR2    of a Nanobody comprises the amino acids at positions 36-49, CDR2 of    a Nanobody comprises the amino acid residues at positions 50-65, FR3    of a Nanobody comprises the amino acid residues at positions 66-94,    CDR3 of a Nanobody comprises the amino acid residues at positions    95-102, and FR4 of a Nanobody comprises the amino acid residues at    positions 103-113.-   x) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, as well as to the prior art mentioned on page 59 of WO08/020,079 and to the list of references mentioned on pages 41-43 of theInternational application WO 06/040153, which prior art and referencesare incorporated herein by reference.

In accordance with the terminology used in the art (see the abovereferences), the variable domains present in naturally occurring heavychain antibodies will also be referred to as “V_(HH) domains”, in orderto distinguish them from the heavy chain variable domains that arepresent in conventional 4-chain antibodies (which will be referred tohereinbelow as “V_(H) domains”) and from the light chain variabledomains that are present in conventional 4-chain antibodies (which willbe referred to hereinbelow as “V_(L) domains”).

As mentioned in the prior art referred to above, V_(HH) domains have anumber of unique structural characteristics and functional propertieswhich make isolated V_(HH) domains (as well as Nanobodies based thereon,which share these structural characteristics and functional propertieswith the naturally occurring V_(HH) domains) and proteins containing thesame highly advantageous for use as functional antigen-binding domainsor proteins. In particular, and without being limited thereto, V_(HH)domains (which have been “designed” by nature to functionally bind to anantigen without the presence of, and without any interaction with, alight chain variable domain) and Nanobodies can function as a single,relatively small, functional antigen-binding structural unit, domain orprotein. This distinguishes the V_(HH) domains from the V_(H) and V_(L)domains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or domains, but need to be combined in some form or another toprovide a functional antigen-binding unit (as in for exampleconventional antibody fragments such as Fab fragments; in ScFv'sfragments, which consist of a V_(H) domain covalently linked to a V_(L)domain).

Because of these unique properties, the use of V_(HH) domains andNanobodies as single antigen-binding proteins or as antigen-bindingdomains (i.e. as part of a larger protein or polypeptide) offers anumber of significant advantages over the use of conventional V_(H) andV_(L) domains, scFv's or conventional antibody fragments (such as Fab-or F(ab′)₂-fragments), including the advantages that are listed on pages60 and 61 of WO 08/020,079.

In a specific and preferred aspect, the invention provides Nanobodiesagainst multiscavenger receptors, and in particular Nanobodies againstmultiscavenger receptors from a warm-blooded animal, and more inparticular Nanobodies against multiscavenger receptors from a mammal,and especially Nanobodies against human multiscavenger receptors; aswell as proteins and/or polypeptides comprising at least one suchNanobody.

In particular, the invention provides Nanobodies against multiscavengerreceptors, and proteins and/or polypeptides comprising the same, thathave improved therapeutic and/or pharmacological properties and/or otheradvantageous properties (such as, for example, improved ease ofpreparation and/or reduced costs of goods), compared to conventionalantibodies against multiscavenger receptors or fragments thereof,compared to constructs that could be based on such conventionalantibodies or antibody fragments (such as Fab′ fragments, F(ab′)₂fragments, ScFv constructs, “diabodies” and other multispecificconstructs (see for example the review by Holliger and Hudson, Nat.Biotechnol. 2005 September; 23(9):1126-36)), and also compared to theso-called “dAb's” or similar (single) domain antibodies that may bederived from variable domains of conventional antibodies. These improvedand advantageous properties will become clear from the furtherdescription herein, and for example include, without limitation, one ormore of:

-   -   increased affinity and/or avidity for multiscavenger receptors,        either in a monovalent format, in a multivalent format (for        example in a bivalent format) and/or in a multispecific format        (for example one of the multispecific formats described        hereinbelow);    -   better suitability for formatting in a multivalent format (for        example in a bivalent format);    -   better suitability for formatting in a multispecific format (for        example one of the multispecific formats described hereinbelow);    -   improved suitability or susceptibility for “humanizing”        substitutions (as defined herein);    -   less immunogenicity, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described hereinbelow);    -   increased stability, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described hereinbelow);    -   increased specificity towards multiscavenger receptors, either        in a monovalent format, in a multivalent format (for example in        a bivalent format) and/or in a multispecific format (for example        one of the multispecific formats described hereinbelow);    -   decreased or where desired increased cross-reactivity with        multiscavenger receptors from different species;        and/or    -   one or more other improved properties desirable for        pharmaceutical use (including prophylactic use and/or        therapeutic use) and/or for diagnostic use (including but not        limited to use for imaging purposes), either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described hereinbelow).

As generally described herein for the amino acid sequences of theinvention, the Nanobodies of the invention are preferably in essentiallyisolated form (as defined herein), or form part of a protein orpolypeptide of the invention (as defined herein), which may comprise oressentially consist of one or more Nanobodies of the invention and whichmay optionally further comprise one or more further amino acid sequences(all optionally linked via one or more suitable linkers). For example,and without limitation, the one or more amino acid sequences of theinvention may be used as a binding unit in such a protein orpolypeptide, which may optionally contain one or more further amino acidsequences that can serve as a binding unit (i.e. against one or moreother targets than multiscavenger receptors), so as to provide amonovalent, multivalent or multispecific polypeptide of the invention,respectively, all as described herein. In particular, such a protein orpolypeptide may comprise or essentially consist of one or moreNanobodies of the invention and optionally one or more (other)Nanobodies (i.e. directed against other targets than multiscavengerreceptors), all optionally linked via one or more suitable linkers, soas to provide a monovalent, multivalent or multispecific Nanobodyconstruct, respectively, as further described herein. Such proteins orpolypeptides may also be in essentially isolated form (as definedherein).

In a Nanobody of the invention, the binding site for binding againstmultiscavenger receptors is preferably formed by the CDR sequences.Optionally, a Nanobody of the invention may also, and in addition to theat least one binding site for binding against multiscavenger receptors,contain one or more further binding sites for binding against otherantigens, proteins or targets. For methods and positions for introducingsuch second binding sites, reference is for example made to Keck andHuston, Biophysical Journal, 71, October 1996, 2002-2011; EP 0 640 130;and WO 06/07260.

As generally described herein for the amino acid sequences of theinvention, when a Nanobody of the invention (or a polypeptide of theinvention comprising the same) is intended for administration to asubject (for example for therapeutic and/or diagnostic purposes asdescribed herein), it is preferably directed against humanmultiscavenger receptors; whereas for veterinary purposes, it ispreferably directed against multiscavenger receptors from the species tobe treated. Also, as with the amino acid sequences of the invention, aNanobody of the invention may or may not be cross-reactive (i.e.directed against multiscavenger receptors from two or more species ofmammal, such as against human multiscavenger receptors andmultiscavenger receptors from at least one of the species of mammalmentioned herein).

Also, again as generally described herein for the amino acid sequencesof the invention, the Nanobodies of the invention may generally bedirected against any antigenic determinant, epitope, part, domain,subunit or confirmation (where applicable) of multiscavenger receptors.

As already described herein, the amino acid sequence and structure of aNanobody can be considered—without however being limited thereto—to becomprised of four framework regions or “FR's” (or sometimes alsoreferred to as “FW's”), which are referred to in the art and herein as“Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as“Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”,respectively; which framework regions are interrupted by threecomplementary determining regions or “CDR's”, which are referred to inthe art as “Complementarity Determining Region 1” or “CDR1”; as“Complementarity Determining Region 2” or “CDR2”; and as“Complementarity Determining Region 3” or “CDR3”, respectively. Somepreferred framework sequences and CDR's (and combinations thereof) thatare present in the Nanobodies of the invention are as described herein.Other suitable CDR sequences can be obtained by the methods describedherein.

According to a non-limiting but preferred aspect of the invention, (theCDR sequences present in) the Nanobodies of the invention are such that:

-   -   the Nanobodies can bind to multiscavenger receptors with a        dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or        less, and preferably 10⁻⁷ to 10¹² moles/liter or less and more        preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association        constant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and        preferably 10² to 10¹² liter/moles or more and more preferably        10⁸ to 10¹² liter/moles);        and/or such that:    -   the Nanobodies can bind to multiscavenger receptors with a        k_(on)-rate of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹,        preferably between 10³ M⁻¹s⁻¹ and 10² M⁻¹s⁻¹, more preferably        between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹        and 10² M⁻¹s⁻¹;        and/or such that they:    -   the Nanobodies can bind to multiscavenger receptors with a        k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹        (providing a near irreversible complex with a t_(1/2) of        multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more        preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴        s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, (the CDR sequences present in) the Nanobodies of theinvention are such that: a monovalent Nanobody of the invention (or apolypeptide that contains only one Nanobody of the invention) ispreferably such that it will bind to multiscavenger receptors with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 500 pM.

The affinity of the Nanobody of the invention against multiscavengerreceptors can be determined in a manner known per se, for example usingthe general techniques for measuring K_(D). K_(A), k_(off) or k_(on)mentioned herein, as well as some of the specific assays describedherein.

Some preferred IC50 values for binding of the Nanobodies of theinvention (and of polypeptides comprising the same) to multiscavengerreceptors will become clear from the further description and examplesherein.

In a preferred but non-limiting aspect, the invention relates to aNanobody (as defined herein) against multiscavenger receptors, whichconsists of 4 framework regions (FR1 to FR4 respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 152-177;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 204-229;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 256-281;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;    or any suitable fragment of such an amino acid sequence.

In particular, according to this preferred but non-limiting aspect, theinvention relates to a Nanobody (as defined herein) againstmultiscavenger receptors, which consists of 4 framework regions (FR1 toFR4 respectively) and 3 complementarity determining regions (CDR1 toCDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 152-177;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    152-177;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 204-229;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    204-229;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 256-281;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;    or any suitable fragment of such an amino acid sequences.

As generally mentioned herein for the amino acid sequences of theinvention, when a Nanobody of the invention contains one or more CDR1sequences according to b) and/or c):

-   i) any amino acid substitution in such a CDR according to b)    and/or c) is preferably, and compared to the corresponding CDR    according to a), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to b) and/or c) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to a);    and/or-   iii) the CDR according to b) and/or c) may be a CDR that is derived    from a CDR according to a) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Similarly, when a Nanobody of the invention contains one or more CDR2sequences according to e) and/or f):

-   i) any amino acid substitution in such a CDR according to e)    and/or f) is preferably, and compared to the corresponding CDR    according to d), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to e) and/or f) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to d);    and/or-   iii) the CDR according to e) and/or f) may be a CDR that is derived    from a CDR according to d) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Also, similarly, when a Nanobody of the invention contains one or moreCDR3 sequences according to h) and/or i):

-   i) any amino acid substitution in such a CDR according to h)    and/or i) is preferably, and compared to the corresponding CDR    according to g), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to h) and/or i) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to g);    and/or-   iii) the CDR according to h) and/or i) may be a CDR that is derived    from a CDR according to g) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

It should be understood that the last three paragraphs generally applyto any Nanobody of the invention that comprises one or more CDR1sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e),f), h) or i), respectively.

Of the Nanobodies of the invention, Nanobodies comprising one or more ofthe CDR's explicitly listed above are particularly preferred; Nanobodiescomprising two or more of the CDR's explicitly listed above are moreparticularly preferred; and Nanobodies comprising three of the CDR'sexplicitly listed above are most particularly preferred.

Some particularly preferred, but non-limiting combinations of CDRsequences, as well as preferred combinations of CDR sequences andframework sequences, are mentioned in Table B-1 below, which lists theCDR sequences and framework sequences that are present in a number ofpreferred (but non-limiting) Nanobodies of the invention. As will beclear to the skilled person, a combination of CDR1, CDR2 and CDR3sequences that occur in the same clone (i.e. CDR1, CDR2 and CDR3sequences that are mentioned on the same line in Table B-1) will usuallybe preferred (although the invention in its broadest sense is notlimited thereto, and also comprises other suitable combinations of theCDR sequences mentioned in Table B-1). Also, a combination of CDRsequences and framework sequences that occur in the same clone (i.e. CDRsequences and framework sequences that are mentioned on the same line inTable B-1) will usually be preferred (although the invention in itsbroadest sense is not limited thereto, and also comprises other suitablecombinations of the CDR sequences and framework sequences mentioned inTable B-1, as well as combinations of such CDR sequences and othersuitable framework sequences, e.g. as further described herein).

Also, in the Nanobodies of the invention that comprise the combinationsof CDR's mentioned in Table B-1, each CDR can be replaced by a CDRchosen from the group consisting of amino acid sequences that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity (as defined herein) withthe mentioned CDR's; in which:

-   i) any amino acid substitution in such a CDR is preferably, and    compared to the corresponding CDR sequence mentioned in Table B-1, a    conservative amino acid substitution (as defined herein);    and/or-   ii) any such CDR sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the corresponding CDR sequence mentioned in Table B-1;    and/or-   iii) any such CDR sequence is a CDR that is derived by means of a    technique for affinity maturation known per se, and in particular    starting from the corresponding CDR sequence mentioned in Table B-1.

However, as will be clear to the skilled person, the (combinations of)CDR sequences, as well as (the combinations of) CDR sequences andframework sequences mentioned in Table B-1 will generally be preferred.

TABLE B-1Preferred combinations of CDR sequences, preferred combinations of framework sequences, and preferred combinations of framework and CDR sequences. (“ID”refers to the SEQ ID NO as used herein and represents sequence listed on the right of the SEQ ID NO) ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4126 EVQLVESG 152 MG 178 WFRQAPGKE 204 AISWSGGRTYYADSVK 230RFTISRENAKNIVYL 256 ENLASSGSAY 282 CGQGT GGLVQAGD REFVA G QMNSLKPEDTAVYCSDDRYNA QVTVSS SLRLSCIAS CAT GRTFT 127 EVQLVESG DRAIG 179 WFRQAPGKE 205CSANNDNRAFYEDSVK 231 RFAVSRDNAKNTVYL 257 RCAAGRVNLY 283 WGKGTL GEVVQPGGREGVA G QMNSLKPEDTAVYY YGMDY VTVSS SLRLSCAAS CAT GFTFD 128 EVQLVESG 154NYAIG 180 WFRQAPGKE 206 CVDRDGGSTYYLDSVT 232 RFTTSRDDAENTVYL 258RLYGCSGYG 284 WGQGT GGLVQPGG REGVS G QMNSLIPDDTAVYYC RDYAD QVTVSSSLRLSCAAS AT GFTLG 129 EVQLVESG 155 TDAFG 181 WFRQAPGKE 207AMRWNGSSSYYADLVK 233 RFTISRDNAKNTVYLL 259 GKRYGYYDY 285 WGQGT GGLVQAGGREFVS G MNSLKPEDTAVYYCT QVTVSS SLRLSCAVS A GRTFS 130 EVQLVESG 156 NYSMG182 WFRQAPGKE 208 TISWSGALTHYTDSVKG 234 RFTISRDNAKNTVYL 260 SDSDYGNKY286 WGQGT GGLVQAGG REFVA QMNSLKPEDTAVYY DY QVTVSS SLRLSCAAS CAA GRTFS131 EVQLVESG 157 DMTMG 183 WFRQAPGKE 209 AISNSGLSTYYQDSVKG 235RFTISRDTANNTVAL 261 RSGWSGQYD 287 WGQGT GGLVQAGG RVFVA QMNSLKPEDTAVYFC YQVTVSS SLRLSCAAS AA GRTVS 132 EVQLVESG 158 NYAMG 184 WFRQAPGKE 210GISWSGDSTLYADSVKG 236 RFTTSRDNAKNTVYL 262 KQGADWAPY 288 WGQGT GGLVQAGGREFVA QMNSLKPEDTANYY DY QVTVSS SLRLSCAAS CAE GRIFN 133 EVQLVESG 159LYRMG 185 WFRQAPGKE 211 AMSTSGAGTYYADSVK 237 RFTISRDNPKNTVYL 263GVRFGVYDY 289 WGQGT GGLVQAGG REFVS G QMNSLKPEDTAVYY QVTVSS SLRLSCVAS CVAELTFS 134 EVQLVESG 160 DYAIG 186 WFRQAPGKE 212 CISRTDGSTDYADSVKG 238RFTISSDNAKNTVYL 264 GRTYYSGSYY 290 WGQGT GGLVQPGG REGVS QMNSLKPEDTAVYYFGLGSDEYDY QVTVSS SLRLSCAAS CAA GFTLD 135 EVQLVESG 161 INAMA 187WYRQAPGKQ 213 HLTNSGRTGYADSVKG 239 RFTISSDNAKNTVYL 265 LGLHWS 291 WGQGTGGLVQPGG RELVA QMNSLKPEDTAVYY QVTVSS SLRLSCAAS CNR GSIFT 136 EVQLVESG162 AYHMG 188 WFRQAPGKE 214 AISWSVSSTYYADSVKG 240 RFTISRDNAKRTVSL 266RSGERYDYYK 292 WGQGT GGLVQAGG RELVA QMDSLKPEDTAVYY AQYEY QVTVSSSLRLSCAAS CAA IGTFS 137 EVQLVESG 163 IGTMG 189 WYRQPPGNQ 215VTYGLGSTNYAESVKG 241 RFTISRDNAKNTVSL 267 EIDTDPRSGE 293 WGQGT GGLVQPGGRELVA QMNSLKPEDTAVYY WDY QVTVSS SLRLSCAAY CYA GSFFS 138 EVQLVESG 164LRTVG 190 WYRQGPGKQ 216 IMSAGTTRYADSVKG 242 RFTISLDDAKNTVYLQ 268RPVFSNVDY 294 WGQGT GGLVQPGG RDLVA MNSLKPEDTAVYICN QVTVSS SLRLSCLPS GTSTSS 139 EVQLVESG 165 DYAIG 191 WFRQAPGKE 217 CVSRDGGSTYYLDSVK 243RFTISSDNAKNTVYL 269 SRYDCSKYLI 295 RGQGT GGLVQAGG REGVS G QMNSLKPEDAAVYYDYNY QVTVSS SLRLSCAAS CAA GFTFD 140 EVQLVKSG 166 TSGMG 192 WFRQAPGRE 218GI?WNSR?TYYAESVKG 244 RFTISRDNSKNTVYL 270 NYYGS?WSV 296 W?QG?Q GGLVQAGGREFV? QMNSLKPEDTAVYY NSDDYDY VTVSS SLRLSCAAS CAT GRRFS 141 EVQLVESG 167NYAMG 193 WFRQAPGKE 219 AITWSGSSTYYADSVKG 245 RFTISRDNAKNTVYL 271AQRGRYYYLD 297 WGQGT GGLVQAGG REFVA QMNSLKPEDTAVYY RNVEYDY QVTVSSSLRLSCAAS CAA GRTFS 142 EVQLVESG 168 DYGIG 194 WFRQAPGKE 220CISSSDGSTDYADSVKG 246 RFTISRDNAKNTVYL 272 GRTYYSGSYY 298 WGQGT GGLVQPGGREGVS QMNNLKPEDTAVYY FGLGSDEYDY QVTVSS SLRLSCAAS CAA GFTLD 143 EVQLVESG169 DYVIG 195 WFRQAPGKE 221 CISSVEGSTYYADSVKG 247 RFTISGDNAKNTVYL 273GTWLDCSGY 299 WGKGTL GNLVQAGG REGVS QMNSLKPEDTAVYY GSYDMDY VTVSSSLRLSCAAS CAA GFTFD 144 EVQLVESG 170 DYVIG 196 WFRQAPGKE 222CISSSEGSTYYAESVKG 248 RFTISSDNAKNTVYL 274 STWLDFVHG 300 RGQGT GGLVQAGGREGVS QMNSLKPEDTAVYY NEYDY QVTVSS SLRLSCAAS CAA GFTFD 145 EVQLVESG 171TYAMG 197 WFRQAPGKE 223 AMISSLSSKSYADTVKG 249 RFTISRDYAKNTVY? 275DLLPYSSSRS 301 WGQGT GGLVQAGG REFVA QMNSLKPEDTADYY LPMGYDY QVTVSSSLRLSCTAS CAA GRAVS 146 EVQLVESG 172 TYAMG 198 WFRQAPGKE 224AMISSLSSKSYADSVKG 250 RFTISRDYAKNTVYL 276 DLLPYSSTRS 302 WGQGT GGLVQAGGREFVA QMNSLKPEDTADYY LPMGYDY QVTVSS SLRLSCTAS CAA GRAVS 147 EVQLVESG 173YDMG 199 WFSQAPGKE 225 AINWSGGSTAYADSVK 251 RFTISRDNAKNTVYL 277KPAKYHFGSG 303 WGQGT GGLVQAGG REFVA G QMNSLKPEDTAVYY YRDFAEYPY QVTVSSSLRLSCAAS CAA GSFSL 148 EVQLVESG 174 RYAMA 200 WFRHAPGKD 226AVSQSGLLTFYADSVKG 252 RFTISRDNAKNTVYL 278 ?SRFPLVVPV 304 WGQGT GGLVQAGGREFVA QMNSLKPEDTAVYD AYEN QVTVSS SLRLSCAAS CAA GRTFS 149 EVQLVESG 175RYAMA 201 WFRHAPGKD 227 AVSQSGLLTFYADSVKG 253 RFTISRDNAKNTVYL 279DSRFPLVVPV 305 WGQGT GGLVQAGG REFVA QMNSLKPEDTAVYD AYEN QVTVSS SLRLSCAASCAA GRTFS 150 EVQLVESG 176 THAMG 202 WYRQAPGKQ 228 TITSVTSGGSLNYADSV 254RFTISRDNAKNTVYL 280 LGFDY 306 RGQGT GGLVQVGG RELVA KG QMNSLKPEDTAVYYQVTVSS SLRLSCAAS CKL GISIR 151 EVQLVESG 177 FVAMG 203 WYRQAPGKQ 229TITSITSGGRTNYADSVK 255 RFTISRDNAKNTVYL 281 VPYVNDY 307 WGQGT GGLVQPGGRELVA G QMNSLKPEDTAVYY QVTVSS SLRLSCAAS CNV GSIGR

Thus, in the Nanobodies of the invention, at least one of the CDR1, CDR2and CDR3 sequences present is suitably chosen from the group consistingof the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1;or from the group of CDR1, CDR2 and CDR3 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% “sequence identity” (as definedherein) with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” (as defined herein) with at least one ofthe CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1.

In this context, by “suitably chosen” is meant that, as applicable, aCDR1 sequence is chosen from suitable CDR1 sequences (i.e. as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. asdefined herein), and a CDR3 sequence is chosen from suitable CDR3sequence (i.e. as defined herein), respectively. More in particular, theCDR sequences are preferably chosen such that the Nanobodies of theinvention bind to multiscavenger receptors with an affinity (suitablymeasured and/or expressed as a K_(D)-value (actual or apparent), aK_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate,or alternatively as an IC₅₀ value, as further described herein) that isas defined herein.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table B-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table B-1; and/or from thegroup consisting of the CDR3 sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the CDR3 sequences listed inTable B-1.

Preferably, in the Nanobodies of the invention, at least two of theCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable B-1 or from the group consisting of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” with at least one of the CDR1, CDR2 andCDR3 sequences, respectively, listed in Table B-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table B-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table B-1, respectively; andat least one of the CDR1 and CDR2 sequences present is suitably chosenfrom the group consisting of the CDR1 and CDR2 sequences, respectively,listed in Table B-1 or from the group of CDR1 and CDR2 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1 and CDR2 sequences, respectively,listed in Table B-1; and/or from the group consisting of the CDR1 andCDR2 sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table B-1.

Most preferably, in the Nanobodies of the invention, all three CDR1,CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable B-1 or from the group of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-1.

Even more preferably, in the Nanobodies of the invention, at least oneof the CDR1, CDR2 and CDR3 sequences present is suitably chosen from thegroup consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table B-1. Preferably, in this aspect, at least one orpreferably both of the other two CDR sequences present are suitablychosen from CDR sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the corresponding CDR sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR sequences that have 3, 2 or only 1 amino acid difference(s) withat least one of the corresponding sequences, respectively, listed inTable B-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table B-1. Preferably, in this aspect, at least one andpreferably both of the CDR1 and CDR2 sequences present are suitablychosen from the groups of CDR1 and CDR2 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with the CDR1and CDR2 sequences, respectively, listed in Table B-1; and/or from thegroup consisting of the CDR1 and CDR2 sequences, respectively, that have3, 2 or only 1 amino acid difference(s) with at least one of the CDR1and CDR2 sequences, respectively, listed in Table B-1.

Even more preferably, in the Nanobodies of the invention, at least twoof the CDR1, CDR2 and CDR3 sequences present are suitably chosen fromthe group consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table B-1. Preferably, in this aspect, the remaining CDRsequence present is suitably chosen from the group of CDR sequences thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with at leastone of the corresponding CDR sequences listed in Table B-1; and/or fromthe group consisting of CDR sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the corresponding sequenceslisted in Table B-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence is suitably chosen from the group consisting of the CDR3sequences listed in Table B-1, and either the CDR1 sequence or the CDR2sequence is suitably chosen from the group consisting of the CDR1 andCDR2 sequences, respectively, listed in Table B-1. Preferably, in thisaspect, the remaining CDR sequence present is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the corresponding CDR sequences listed inTable B-1; and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with the corresponding CDRsequences listed in Table B-1.

Even more preferably, in the Nanobodies of the invention, all threeCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable B-1.

Also, generally, the combinations of CDR's listed in Table B-1 (i.e.those mentioned on the same line in Table B-1) are preferred. Thus, itis generally preferred that, when a CDR in a Nanobody of the inventionis a CDR sequence mentioned in Table B-1 or is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with a CDR sequence listed in Table B-1; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with a CDR sequence listed in Table B-1, that at least oneand preferably both of the other CDR's are suitably chosen from the CDRsequences that belong to the same combination in Table B-1 (i.e.mentioned on the same line in Table B-1) or are suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with the CDR sequence(s) belonging to the same combinationand/or from the group consisting of CDR sequences that have 3, 2 or only1 amino acid difference(s) with the CDR sequence(s) belonging to thesame combination. The other preferences indicated in the aboveparagraphs also apply to the combinations of CDR's mentioned in TableB-1.

Thus, by means of non-limiting examples, a Nanobody of the invention canfor example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table B-1, a CDR2sequence that has 3, 2 or 1 amino acid difference with one of the CDR2sequences mentioned in Table B-1 (but belonging to a differentcombination), and a CDR3 sequence.

Some preferred Nanobodies of the invention may for example comprise: (1)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table B-1; a CDR2 sequence that has 3, 2 or1 amino acid difference with one of the CDR2 sequences mentioned inTable B-1 (but belonging to a different combination); and a CDR3sequence that has more than 80% sequence identity with one of the CDR3sequences mentioned in Table B-1 (but belonging to a differentcombination); or (2) a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table B-1; a CDR2sequence, and one of the CDR3 sequences listed in Table B-1; or (3) aCDR1 sequence; a CDR2 sequence that has more than 80% sequence identitywith one of the CDR2 sequence listed in Table B-1; and a CDR3 sequencethat has 3, 2 or 1 amino acid differences with the CDR3 sequencementioned in Table B-1 that belongs to the same combination as the CDR2sequence.

Some particularly preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table B-1; a CDR2 sequencethat has 3, 2 or 1 amino acid difference with the CDR2 sequencementioned in Table B-1 that belongs to the same combination; and a CDR3sequence that has more than 80% sequence identity with the CDR3 sequencementioned in Table B-1 that belongs to the same combination; (2) a CDR1sequence; a CDR 2 listed in Table B-1 and a CDR3 sequence listed inTable B-1 (in which the CDR2 sequence and CDR3 sequence may belong todifferent combinations).

Some even more preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table B-1; the CDR2 sequencelisted in Table B-1 that belongs to the same combination; and a CDR3sequence mentioned in Table B-1 that belongs to a different combination;or (2) a CDR1 sequence mentioned in Table B-1; a CDR2 sequence that has3, 2 or 1 amino acid differences with the CDR2 sequence mentioned inTable B-1 that belongs to the same combination; and a CDR3 sequence thathas more than 80% sequence identity with the CDR3 sequence listed inTable B-1 that belongs to the same or a different combination.

Particularly preferred Nanobodies of the invention may for examplecomprise a CDR1 sequence mentioned in Table B-1, a CDR2 sequence thathas more than 80% sequence identity with the CDR2 sequence mentioned inTable B-1 that belongs to the same combination; and the CDR3 sequencementioned in Table B-1 that belongs to the same combination.

In the most preferred Nanobodies of the invention, the CDR1, CDR2 andCDR3 sequences present are suitably chosen from one of the combinationsof CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1.

According to another preferred, but non-limiting aspect of the invention(a) CDR1 has a length of between 1 and 12 amino acid residues, andusually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues; and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences (as defined herein) have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more sequence identity (as defined herein) with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 308-333 (seeTable A-1).

Generally, Nanobodies with the above CDR sequences may be as furtherdescribed herein, and preferably have framework sequences that are alsoas further described herein. Thus, for example and as mentioned herein,such Nanobodies may be naturally occurring Nanobodies (from any suitablespecies), naturally occurring V_(HH) sequences (i.e. from a suitablespecies of Camelid) or synthetic or semi-synthetic amino acid sequencesor Nanobodies, including but not limited to partially humanizedNanobodies or V_(HH) sequences, fully humanized Nanobodies or V_(HH)sequences, camelized heavy chain variable domain sequences, as well asNanobodies that have been obtained by the techniques mentioned herein.

Thus, in one specific, but non-limiting aspect, the invention relates toa humanized Nanobody, which consists of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which CDR1 to CDR3 are as defined herein and in whichsaid humanized Nanobody comprises at least one humanizing substitution(as defined herein), and in particular at least one humanizingsubstitution in at least one of its framework sequences (as definedherein).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 308-333 (seeTable A-1). This degree of amino acid identity can for example bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said Nanobody and one or more of the sequencesof SEQ ID NO's: 308-333 (see Table A-1), in which the amino acidresidues that form the framework regions are disregarded. SuchNanobodies can be as further described herein.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody with an amino acid sequence that is chosen from the groupconsisting of SEQ ID NO's: 308-333 (see Table A-1) or from the groupconsisting of from amino acid sequences that have more than 80%,preferably more than 90%, more preferably more than 95%, such as 99% ormore sequence identity (as defined herein) with at least one of theamino acid sequences of SEQ ID NO's: 308-333 (see Table A-1).

Another preferred, but non-limiting aspect of the invention relates tohumanized variants of the Nanobodies of SEQ ID NO's: 308-333 (see TableA-1), that comprise, compared to the corresponding native V_(HH)sequence, at least one humanizing substitution (as defined herein), andin particular at least one humanizing substitution in at least one ofits framework sequences (as defined herein).

It will be clear to the skilled person that the Nanobodies that arementioned herein as “preferred” (or “more preferred”, “even morepreferred”, etc.) are also preferred (or more preferred, or even morepreferred, etc.) for use in the polypeptides described herein. Thus,polypeptides that comprise or essentially consist of one or more“preferred” Nanobodies of the invention will generally be preferred, andpolypeptides that comprise or essentially consist of one or more “morepreferred” Nanobodies of the invention will generally be more preferred,etc.

Generally, proteins or polypeptides that comprise or essentially consistof a single Nanobody (such as a single Nanobody of the invention) willbe referred to herein as “monovalent” proteins or polypeptides or as“monovalent constructs”. Proteins and polypeptides that comprise oressentially consist of two or more Nanobodies (such as at least twoNanobodies of the invention or at least one Nanobody of the inventionand at least one other Nanobody) will be referred to herein as“multivalent” proteins or polypeptides or as “multivalent constructs”,and these may provide certain advantages compared to the correspondingmonovalent Nanobodies of the invention. Some non-limiting examples ofsuch multivalent constructs will become clear from the furtherdescription herein.

According to one specific, but non-limiting aspect, a polypeptide of theinvention comprises or essentially consists of at least two Nanobodiesof the invention, such as two or three Nanobodies of the invention. Asfurther described herein, such multivalent constructs can providecertain advantages compared to a protein or polypeptide comprising oressentially consisting of a single Nanobody of the invention, such as amuch improved avidity for multiscavenger receptors.

According to another specific, but non-limiting aspect, a polypeptide ofthe invention comprises or essentially consists of at least one Nanobodyof the invention and at least one other binding unit (i.e. directedagainst another epitope, antigen, target, protein or polypeptide), whichis preferably also a Nanobody. Such proteins or polypeptides are alsoreferred to herein as “multispecific” proteins or polypeptides or as“multispecific constructs”, and these may provide certain advantagescompared to the corresponding monovalent Nanobodies of the invention (aswill become clear from the further discussion herein of some preferred,but-nonlimiting multispecific constructs).

According to yet another specific; but non-limiting aspect, apolypeptide of the invention comprises or essentially consists of atleast one Nanobody of the invention, optionally one or more furtherNanobodies, and at least one other amino acid sequence (such as aprotein or polypeptide) that confers at least one desired property tothe Nanobody of the invention and/or to the resulting fusion protein.Again, such fusion proteins may provide certain advantages compared tothe corresponding monovalent Nanobodies of the invention. Somenon-limiting examples of such amino acid sequences and of such fusionconstructs will become clear from the further description herein.

It is also possible to combine two or more of the above aspects, forexample to provide a trivalent bispecific construct comprising twoNanobodies of the invention and one other Nanobody, and optionally oneor more other amino acid sequences. Further non-limiting examples ofsuch constructs, as well as some constructs that are particularlypreferred within the context of the present invention, will become clearfrom the further description herein.

In the above constructs, the one or more Nanobodies and/or other aminoacid sequences may be directly linked to each other and/or suitablylinked to each other via one or more linker sequences. Some suitable butnon-limiting examples of such linkers will become clear from the furtherdescription herein.

In one specific aspect of the invention, a Nanobody of the invention ora compound, construct or polypeptide of the invention comprising atleast one Nanobody of the invention may have an increased half-life,compared to the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such Nanobodies, compounds andpolypeptides will become clear to the skilled person based on thefurther disclosure herein, and for example comprise Nanobodies sequencesor polypeptides of the invention that have been chemically modified toincrease the half-life thereof (for example, by means of pegylation);amino acid sequences of the invention that comprise at least oneadditional binding site for binding to a serum protein (such as serumalbumin, see for example EP 0 368 684 B1, page 4); or polypeptides ofthe invention that comprise at least one Nanobody of the invention thatis linked to at least one moiety (and in particular at least one aminoacid sequence) that increases the half-life of the Nanobody of theinvention. Examples of polypeptides of the invention that comprise suchhalf-life extending moieties or amino acid sequences will become clearto the skilled person based on the further disclosure herein; and forexample include, without limitation, polypeptides in which the one ormore Nanobodies of the invention are suitable linked to one or moreserum proteins or fragments thereof (such as serum albumin or suitablefragments thereof) or to one or more binding units that can bind toserum proteins (such as, for example, Nanobodies or (single) domainantibodies that can bind to serum proteins such as serum albumin, serumimmunoglobulins such as IgG, or transferrine); polypeptides in which aNanobody of the invention is linked to an Fc portion (such as a humanFc) or a suitable part or fragment thereof; or polypeptides in which theone or more Nanobodies of the invention are suitable linked to one ormore small proteins or peptides that can bind to serum proteins (suchas, without limitation, the proteins and peptides described in WO91/01743, WO 01/45746, WO 02/076489 and to the US provisionalapplication of Ablynx N.V. entitled “Peptides capable of binding toserum proteins” of Ablynx N.V. filed on Dec. 5, 2006 (see alsoPCT/EP/2007/063348).

Again, as will be clear to the skilled person, such Nanobodies,compounds, constructs or polypeptides may contain one or more additionalgroups, residues, moieties or binding units, such as one or more furtheramino acid sequences and in particular one or more additional Nanobodies(i.e. not directed against multiscavenger receptors), so as to provide atri- of multi specific Nanobody construct.

Generally, the Nanobodies of the invention (or compounds, constructs orpolypeptides comprising the same) with increased half-life preferablyhave a half-life that is at least 1.5 times, preferably at least 2times, such as at least 5 times, for example at least 10 times or morethan 20 times, greater than the half-life of the corresponding aminoacid sequence of the invention per se. For example, the Nanobodies,compounds, constructs or polypeptides of the invention with increasedhalf-life may have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchNanobodies, compound, constructs or polypeptides of the inventionexhibit a serum half-life in human of at least about 12 hours,preferably at least 24 hours, more preferably at least 48 hours, evenmore preferably at least 72 hours or more. For example, compounds orpolypeptides of the invention may have a half-life of at least 5 days(such as about 5 to 10 days), preferably at least 9 days (such as about9 to 14 days), more preferably at least about 10 days (such as about 10to 15 days), or at least about 11 days (such as about 11 to 16 days),more preferably at least about 12 days (such as about 12 to 18 days ormore), or more than 14 days (such as about 14 to 19 days).

In another one aspect of the invention, a polypeptide of the inventioncomprises one or more (such as two or preferably one) Nanobodies of theinvention linked (optionally via one or more suitable linker sequences)to one or more (such as two and preferably one) amino acid sequencesthat allow the resulting polypeptide of the invention to cross the bloodbrain barrier. In particular, said one or more amino acid sequences thatallow the resulting polypeptides of the invention to cross the bloodbrain barrier may be one or more (such as two and preferably one)Nanobodies, such as the Nanobodies described in WO 02/057445, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In particular, polypeptides comprising one or more Nanobodies of theinvention are preferably such that they:

-   -   bind to multiscavenger receptors with a dissociation constant        (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably        10⁻⁷ to 10⁻¹² moles/liter or less and more preferably 10⁻⁸ to        10⁻¹² moles/liter (i.e. with an association constant (K_(A)) of        10⁵ to 10¹² liter/moles or more, and preferably 10⁷ to 10¹²        liter/moles or more and more preferably 10⁸ to 10¹²        liter/moles);        and/or such that they:    -   bind to multiscavenger receptors with a k_(on)-rate of between        10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10⁻³ M⁻¹s⁻¹        and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷        M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to multiscavenger receptors with a k_(off) rate between 1        s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶        s⁻¹, such as between 10⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a polypeptide that contains only one amino acid sequence ofthe invention is preferably such that it will bind to multiscavengerreceptors with an affinity less than 500 nM, preferably less than 200nM, more preferably less than 10 nM, such as less than 500 pM. In thisrespect, it will be clear to the skilled person that a polypeptide thatcontains two or more Nanobodies of the invention may bind tomultiscavenger receptors with an increased avidity, compared to apolypeptide that contains only one amino acid sequence of the invention.

Some preferred IC₅₀ values for binding of the amino acid sequences orpolypeptides of the invention to multiscavenger receptors will becomeclear from the further description and examples herein.

Another aspect of this invention relates to a nucleic acid that encodesan amino acid sequence of the invention (such as a Nanobody of theinvention) or a polypeptide of the invention comprising the same. Again,as generally described herein for the nucleic acids of the invention,such a nucleic acid may be in the form of a genetic construct, asdefined herein.

In another aspect, the invention relates to host or host cell thatexpresses or that is capable of expressing an amino acid sequence (suchas a Nanobody) of the invention and/or a polypeptide of the inventioncomprising the same; and/or that contains a nucleic acid of theinvention. Some preferred but non-limiting examples of such hosts orhost cells will become clear from the further description herein.

Another aspect of the invention relates to a product or compositioncontaining or comprising at least one amino acid sequence of theinvention, at least one polypeptide of the invention and/or at least onenucleic acid of the invention, and optionally one or more furthercomponents of such compositions known per se, i.e. depending on theintended use of the composition. Such a product or composition may forexample be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention further relates to methods for preparing or generating theamino acid sequences, compounds, constructs, polypeptides, nucleicacids, host cells, products and compositions described herein. Somepreferred but non-limiting examples of such methods will become clearfrom the further description herein.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with multiscavenger receptors. Some preferred butnon-limiting applications and uses will become clear from the furtherdescription herein.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description hereinbelow.

Generally, it should be noted that the term Nanobody as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the Nanobodies of the invention can generally beobtained by any of the techniques (1) to (8) mentioned on pages 61 and62 of WO 08/020,079, or any other suitable technique known per se. Onepreferred class of Nanobodies corresponds to the V_(HH) domains ofnaturally occurring heavy chain antibodies directed againstmultiscavenger receptors. As further described herein, such V_(HH)sequences can generally be generated or obtained by suitably immunizinga species of Camelid with multiscavenger receptors (i.e. so as to raisean immune response and/or heavy chain antibodies directed againstmultiscavenger receptors), by obtaining a suitable biological samplefrom said Camelid (such as a blood sample, serum sample or sample ofB-cells), and by generating V_(HH) sequences directed againstmultiscavenger receptors, starting from said sample, using any suitabletechnique known per se. Such techniques will be clear to the skilledperson and/or are further described herein.

Alternatively, such naturally occurring V_(HH) domains againstmultiscavenger receptors, can be obtained from naïve libraries ofCamelid V_(HH) sequences, for example by screening such a library usingmultiscavenger receptors, or at least one part, fragment, antigenicdeterminant or epitope thereof using one or more screening techniquesknown per se. Such libraries and techniques are for example described inWO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively,improved synthetic or semi-synthetic libraries derived from naïve V_(HH)libraries may be used, such as V_(HH) libraries obtained from naïveV_(HH) libraries by techniques such as random mutagenesis and/or CDRshuffling, as for example described in WO 00/43507.

Thus, in another aspect, the invention relates to a method forgenerating Nanobodies, that are directed against multiscavengerreceptors. In one aspect, said method at least comprises the steps of:

-   a) providing a set, collection or library of Nanobody sequences; and-   b) screening said set, collection or library of Nanobody sequences    for Nanobody sequences that can bind to and/or have affinity for    multiscavenger receptors;    and-   c) isolating the Nanobody or Nanobodies that can bind to and/or have    affinity for multiscavenger receptors.

In such a method, the set, collection or library of Nanobody sequencesmay be a naïve set, collection or library of Nanobody sequences; asynthetic or semi-synthetic set, collection or library of Nanobodysequences; and/or a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofNanobody sequences may be an immune set, collection or library ofNanobody sequences, and in particular an immune set, collection orlibrary of V_(HH) sequences, that have been derived from a species ofCamelid that has been suitably immunized with multiscavenger receptorsor with a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

In the above methods, the set, collection or library of Nanobody orV_(HH) sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-omanism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) Nanobody sequences will beclear to the person skilled in the art, for example on the basis of thefurther disclosure herein, Reference is also made to WO 03/054016 and tothe review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116(2005).

In another aspect, the method for generating Nanobody sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells derived from a species    of Camelid that express immunoglobulin sequences;-   b) screening said collection or sample of cells for (i) cells that    express an immunoglobulin sequence that can bind to and/or have    affinity for multiscavenger receptors; and (ii) cells that express    heavy chain antibodies, in which substeps (i) and (ii) can be    performed essentially as a single screening step or in any suitable    order as two separate screening steps, so as to provide at least one    cell that expresses a heavy chain antibody that can bind to and/or    has affinity for multiscavenger receptors;    and-   c) either (i) isolating from said cell the V_(HH) sequence present    in said heavy chain antibody; or (ii) isolating from said cell a    nucleic acid sequence that encodes the V_(HH) sequence present in    said heavy chain antibody, followed by expressing said V_(HH)    domain.

In the method according to this aspect, the collection or sample ofcells may for example be a collection or sample of B-cells. Also, inthis method, the sample of cells may be derived from a Camelid that hasbeen suitably immunized with multiscavenger receptors or a suitableantigenic determinant based thereon or derived therefrom, such as anantigenic part, fragment, region, domain, loop or other epitope thereof.In one particular aspect, said antigenic determinant may be anextracellular part, region, domain, loop or other extracellularepitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood.Vol. 97, No. 12, 3820. Particular reference is made to the so-called“Nanoclone™” technique described in International application WO06/079372 by Ablynx N.V.

In another aspect, the method for generating an amino acid sequencedirected against multiscavenger receptors may comprise at least thesteps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding heavy chain antibodies or Nanobody sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode a heavy chain    antibody or a Nanobody sequence that can bind to and/or has affinity    for multiscavenger receptors;    and-   c) isolating said nucleic acid sequence, followed by expressing the    V_(HH) sequence present in said heavy chain antibody or by    expressing said Nanobody sequence, respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding heavy chain antibodies or Nanobody sequences may forexample be a set, collection or library of nucleic acid sequencesencoding a naïve set, collection or library of heavy chain antibodies orV_(HH) sequences; a set, collection or library of nucleic acid sequencesencoding a synthetic or semi-synthetic set, collection or library ofNanobody sequences; and/or a set, collection or library of nucleic acidsequences encoding a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences encoding heavy chain antibodies or V_(HH)sequences derived from a Camelid that has been suitably immunized withmultiscavenger receptors or with a suitable antigenic determinant basedthereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to WO 03/054016 and to the review by Hoogenboomin Nature Biotechnology, 23, 9, 1105-1116 (2005).

As will be clear to the skilled person, the screening step of themethods described herein can also be performed as a selection step.Accordingly the term “screening” as used in the present description cancomprise selection, screening or any suitable combination of selectionand/or screening techniques. Also, when a set, collection or library ofsequences is used, it may contain any suitable number of sequences, suchas 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷,10⁸ or more sequences.

Also, one or more or all of the sequences in the above set, collectionor library of amino acid sequences may be obtained or defined byrational, or semi-empirical approaches such as computer modellingtechniques or biostatics or datamining techniques.

Furthermore, such a set, collection or library can comprise one, two ormore sequences that are variants from one another (e.g. with designedpoint mutations or with randomized positions), compromise multiplesequences derived from a diverse set of naturally diversified sequences(e.g. an immune library)), or any other source of diverse sequences (asdescribed for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005and Binz et al, Nat Biotechnol 2005, 23:1247). Such set, collection orlibrary of sequences can be displayed on the surface of a phageparticle, a ribosome, a bacterium, a yeast cell, a mammalian cell, andlinked to the nucleotide sequence encoding the amino acid sequencewithin these carriers. This makes such set, collection or libraryamenable to selection procedures to isolate the desired amino acidsequences of the invention. More generally, when a sequence is displayedon a suitable host or host cell, it is also possible (and customary) tofirst isolate from said host or host cell a nucleotide sequence thatencodes the desired sequence, and then to obtain the desired sequence bysuitably expressing said nucleotide sequence in a suitable hostorganism. Again, this can be performed in any suitable manner known perse, as will be clear to the skilled person.

Yet another technique for obtaining V_(HH) sequences or Nanobodysequences directed against multiscavenger receptors involves suitablyimmunizing a transgenic mammal that is capable of expressing heavy chainantibodies (i.e. so as to raise an immune response and/or heavy chainantibodies directed against multiscavenger receptors), obtaining asuitable biological sample from said transgenic mammal that contains(nucleic acid sequences encoding) said V_(HH) sequences or Nanobodysequences (such as a blood sample, serum sample or sample of B-cells),and then generating V_(HH) sequences directed against multiscavengerreceptors, starting from said sample, using any suitable technique knownper se (such as any of the methods described herein or a hybridomatechnique). For example, for this purpose, the heavy chainantibody-expressing mice and the further methods and techniquesdescribed in WO 02/085945, WO 04/049794 and WO 06/008548 and Janssens etal., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10; 103(41):15130-5 can beused. For example, such heavy chain antibody expressing mice can expressheavy chain antibodies with any suitable (single) variable domain, suchas (single) variable domains from natural sources (e.g. human (single)variable domains, Camelid (single) variable domains or shark (single)variable domains), as well as for example synthetic or semi-synthetic(single) variable domains.

The invention also relates to the V_(HH) sequences or Nanobody sequencesthat are obtained by the above methods, or alternatively by a methodthat comprises the one of the above methods and in addition at least thesteps of determining the nucleotide sequence or amino acid sequence ofsaid V_(HH) sequence or Nanobody sequence; and of expressing orsynthesizing said. V_(HH) sequence or Nanobody sequence in a mannerknown per se, such as by expression in a suitable host cell or hostorganism or by chemical synthesis.

As mentioned herein, a particularly preferred class of Nanobodies of theinvention comprises Nanobodies with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring V_(HH)domain, but that has been “humanized”, i.e. by replacing one or moreamino acid residues in the amino acid sequence of said naturallyoccurring V_(HH) sequence (and in particular in the framework sequences)by one or more of the amino acid residues that occur at thecorresponding position(s) in a V_(H) domain from a conventional 4-chainantibody from a human being (e.g. indicated above), as further describedon, and using the techniques mentioned on, page 63 of WO 08/020,079.Another particularly preferred class of Nanobodies of the inventioncomprises Nanobodies with an amino acid sequence that corresponds to theamino acid sequence of a naturally occurring V_(H) domain, but that hasbeen “camelized”, i.e. by replacing one or more amino acid residues inthe amino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody, as further described on, and using the techniquesmentioned on, page 63 of WO 08/020,079.

Other suitable methods and techniques for obtaining the Nanobodies ofthe invention and/or nucleic acids encoding the same, starting fromnaturally occurring V_(H) sequences or preferably V_(HH) sequences, willbe clear from the skilled person, and may for example include thetechniques that are mentioned on page 64 of WO 08/00279 As mentionedherein, Nanobodies may in particular be characterized by the presence ofone or more “Hallmark residues” (as described herein) in one or more ofthe framework sequences.

Thus, according to one preferred, but non-limiting aspect of theinvention, a Nanobody in its broadest sense can be generally defined asa polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 45    according to the Kabat numbering is a charged amino acid (as defined    herein) or a cysteine residue, and position 44 is preferably an E;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of P, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:-   b) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid or a cysteine and the amino acid    residue at position 44 according to the Kabat numbering is    preferably E;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a Nanobody in its broadest sense can be generally definedas a polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 108    according to the Kabat numbering is Q;    and/or:-   b) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 44    according to the Kabat numbering is E and in which the amino acid    residue at position 45 according to the Kabat numbering is an R;    and/or:-   c) an amino acid sequence that is comprised of four framework    regions/sequences interrupted by three complementarity determining    regions/sequences, in which the amino acid residue at position 103    according to the Kabat numbering is chosen from the group consisting    of F, R and S, and is in particular chosen from the group consisting    of R and S.

Thus, according to a preferred, but non-limiting aspect, a Nanobody ofthe invention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, a Nanobody against multiscavenger receptors according tothe invention may have the structure:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which

-   a) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and/or in which:-   b) the amino acid residue at position 44 according to the Kabat    numbering is E and in which the amino acid residue at position 45    according to the Kabat numbering is an R;    and/or in which:-   c) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S, and is    in particular chosen from the group consisting of R and S;    and in which:-   d) CDR1. CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In particular, according to one preferred, but non-limiting aspect ofthe invention, a Nanobody can generally be defined as a polypeptidecomprising an amino acid sequence that is comprised of four frameworkregions/sequences interrupted by three complementarity determiningregions/sequences, in which;

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q; and-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R; and-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    or in which:-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q; and-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R; and-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    or in which:-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q; and-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R; and-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S; and-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   a-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, G, Q,    R, S, L; and is preferably chosen from the group consisting of G, E    or Q;    and in which:-   a-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R or C; and is    preferably chosen from the group consisting of L or R;    and in which:-   a-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R or S; and is    preferably W or R, and is most preferably W;    and in which-   a-4) the amino acid residue at position 108 according to the Kabat    numbering is Q;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   b-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of E and Q;    and in which:-   b-2) the amino acid residue at position 45 according to the Kabat    numbering is R;    and in which:-   b-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of W, R and S; and is    preferably W;    and in which:-   b-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; and is    preferably Q;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   c-1) the amino acid residue at position 44 according to the Kabat    numbering is chosen from the group consisting of A, G, E, D, Q, R, S    and L; and is preferably chosen from the group consisting of G, E    and Q;    and in which:-   c-2) the amino acid residue at position 45 according to the Kabat    numbering is chosen from the group consisting of L, R and C; and is    preferably chosen from the group consisting of L and R;    and in which:-   c-3) the amino acid residue at position 103 according to the Kabat    numbering is chosen from the group consisting of P, R and S; and is    in particular chosen from the group consisting of R and S;    and in which:-   c-4) the amino acid residue at position 108 according to the Kabat    numbering is chosen from the group consisting of Q and L; is    preferably Q;    and in which:-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

Two particularly preferred, but non-limiting groups of the Nanobodies ofthe invention are those according to a) above; according to (a-1) to(a-4) above; according to b) above; according to (b-1) to (b-4) above;according to (c) above; and/or according to (c-1) to (c-4) above, inwhich either:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence CLEW (or a CLEW-like sequence as    described herein) and the amino acid residue at position 108 is Q;    or in which:-   ii) the amino acid residues at positions 43-46 according to the    Kabat numbering form the sequence KERE or KQRE (or a KERE-like    sequence as described) and the amino acid residue at position 108 is    Q or L, and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the amino acid residues at positions 44-47 according to the Kabat    numbering form the sequence GLEW (or a GLEW-like sequence as defined    herein) and the amino acid residue at position 108 is Q;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may have the structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) the amino acid residues at positions 43-46 according to the Kabat    numbering form the sequence KERE or KQRE (or a KERE-like sequence)    and the amino acid residue at position 108 is Q or L, and is    preferably Q;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the Nanobodies of the invention in which the amino acid residues atpositions 43-46 according to the Kabat numbering form the sequence KEREor KQRE, the amino acid residue at position 37 is most preferably F. Inthe Nanobodies of the invention in which the amino acid residues atpositions 44-47 according to the Kabat numbering form the sequence GLEW,the amino acid residue at position 37 is chosen from the groupconsisting of Y, H, I, L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the aminoacid residues present on the positions mentioned above, the Nanobodiesof the invention can generally be classified on the basis of thefollowing three groups:

-   i) The “GLEW-group”: Nanobodies with the amino acid sequence GLEW at    positions 44-47 according to the Kabat numbering and Q at position    108 according to the Kabat numbering. As further described herein,    Nanobodies within this group usually have a V at position 37, and    can have a W, P, R or S at position 103, and preferably have a W at    position 103. The GLEW group also comprises some GLEW-like sequences    such as those mentioned in Table B-2 below. More generally, and    without limitation, Nanobodies belonging to the GLEW-group can be    defined as Nanobodies with a G at position 44 and/or with a W at    position 47, in which position 46 is usually E and in which    preferably position 45 is not a charged amino acid residue and not    cysteine;-   ii) The “KERE-group”: Nanobodies with the amino acid sequence KERE    or KQRE (or another KERE-like sequence) at positions 43-46 according    to the Kabat numbering and Q or L at position 108 according to the    Kabat numbering. As further described herein, Nanobodies within this    group usually have a F at position 37, an L or F at position 47; and    can have a W, P, R or S at position 103, and preferably have a W at    position 103. More generally, and without limitation, Nanobodies    belonging to the KERE-group can be defined as Nanobodies with a K, Q    or R at position 44 (usually K) in which position 45 is a charged    amino acid residue or cysteine, and position 47 is as further    defined herein;-   iii) The “103 P, R, S-group”: Nanobodies with a P, R or S at    position 103. These Nanobodies can have either the amino acid    sequence GLEW at positions 44-47 according to the Kabat numbering or    the amino acid sequence KERE or KQRE at positions 43-46 according to    the Kabat numbering, the latter most preferably in combination with    an F at position 37 and an L or an F at position 47 (as defined for    the KERE-group); and can have Q or L at position 108 according to    the Kabat numbering, and preferably have Q.

Also, where appropriate, Nanobodies may belong to (i.e. havecharacteristics 00 two or more of these classes. For example, onespecifically preferred group of Nanobodies has GLEW or a GLEW-likesequence at positions 44-47; P, R or S (and in particular R) at position103; and Q at position 108 (which may be humanized to L).

More generally, it should be noted that the definitions referred toabove describe and apply to Nanobodies in the form of a native (i.e.non-humanized) V_(HH) sequence, and that humanized variants of theseNanobodies may contain other amino acid residues than those indicatedabove (i.e. one or more humanizing substitutions as defined herein). Forexample, and without limitation, in some humanized Nanobodies of theGLEW-group or the 103 P, R, S-group, Q at position 108 may be humanizedto 108L. As already mentioned herein, other humanizing substitutions(and suitable combinations thereof) will become clear to the skilledperson based on the disclosure herein. In addition, or alternatively,other potentially useful humanizing substitutions can be ascertained bycomparing the sequence of the framework regions of a naturally occurringV_(HH) sequence with the corresponding framework sequence of one or moreclosely related human V₁₄ sequences, after which one or more of thepotentially useful humanizing substitutions (or combinations thereof)thus determined can be introduced into said V_(HH) sequence (in anymanner known per se, as further described herein) and the resultinghumanized V_(HH) sequences can be tested for affinity for the target,for stability, for ease and level of expression, and/or for otherdesired properties. In this way, by means of a limited degree of trialand error, other suitable humanizing substitutions (or suitablecombinations thereof) can be determined by the skilled person based onthe disclosure herein. Also, based on the foregoing, (the frameworkregions of) a Nanobody may be partially humanized or fully humanized.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the GLEW-group (as definedherein), and in which CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the KERE-group (as definedherein), and CDR1, CDR2 and CDR3 are as defined herein, and arepreferably as defined according to one of the preferred aspects herein,and are more preferably as defined according to one of the morepreferred aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of theinvention may be a Nanobody belonging to the 103 P, R, S-group (asdefined herein), and in which CDR1, CDR2 and CDR3 are as defined herein,and are preferably as defined according to one of the preferred aspectsherein, and are more preferably as defined according to one of the morepreferred aspects herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P, R,S residues mentioned above, the Nanobodies of the invention can contain,at one or more positions that in a conventional V₁₄ domain would form(part of) the V_(H)/V_(L) interface, one or more amino acid residuesthat are more highly charged than the amino acid residues that naturallyoccur at the same position(s) in the corresponding naturally occurringV_(H) sequence, and in particular one or more charged amino acidresidues (as mentioned in Table A-2 on page 48 of the Internationalapplication WO 08/020,079). Such substitutions include, but are notlimited to, the GLEW-like sequences mentioned in Table B-2 below; aswell as the substitutions that are described in the InternationalApplication WO 00/29004 for so-called “microbodies”, e.g. so as toobtain a Nanobody with Q at position 108 in combination with KLEW atpositions 44-47. Other possible substitutions at these positions will beclear to the skilled person based upon the disclosure herein.

In one aspect of the Nanobodies of the invention, the amino acid residueat position 83 is chosen from the group consisting of L, M, S, V and W;and is preferably L.

Also, in one aspect of the Nanobodies of the invention, the amino acidresidue at position 83 is chosen from the group consisting of R, K, N,E, G, I, T and Q; and is most preferably either K or E (for Nanobodiescorresponding to naturally occurring V_(HH) domains) or R (for“humanized” Nanobodies, as described herein). The amino acid residue atposition 84 is chosen from the group consisting of P, A, R, S, D T, andV in one aspect, and is most preferably P (for Nanobodies correspondingto naturally occurring V_(HH) domains) or R (for “humanized” Nanobodies,as described herein).

Furthermore, in one aspect of the Nanobodies of the invention, the aminoacid residue at position 104 is chosen from the group consisting of Gand D; and is most preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108, which in the Nanobodies are as mentionedabove, will also be referred to herein as the “Hallmark Residues”. TheHallmark Residues and the amino acid residues at the correspondingpositions of the most closely related human V_(H) domain, V_(H)3, aresummarized in Table B-2.

Some especially preferred but non-limiting combinations of theseHallmark Residues as occur in naturally occurring V_(HH) domains arementioned in Table B-3. For comparison, the corresponding amino acidresidues of the human V_(H)3 called DP-47 have been indicated initalics.

TABLE B-2 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues  11 L, V; L, S, V, M, W, F, T, Q, E, A, R, G, K, Y,predominantly L N, P, I; preferably L  37 V, I, F; usually V F⁽¹⁾, Y, V,L, A, H, S, I, W, C, N, G, D, T, P, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G E⁽³⁾,Q⁽³⁾, G⁽²⁾, D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G⁽²⁾,E⁽³⁾ or Q⁽³⁾; most preferably G⁽²⁾ or Q⁽³⁾  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, P, H, F,G, Q, S, E, T, Y, C, I, D, V; preferably L⁽²⁾ or R⁽³⁾  47⁽⁸⁾ W, Y F⁽¹⁾,L⁽¹⁾ or W⁽²⁾ G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D;preferably W⁽²⁾, L⁽¹⁾ or F⁽¹⁾  83 R or K; usually R R, K⁽⁵⁾, T, E⁽⁵⁾, Q,N, S, I, V, G, M, L, A, D, Y, H; preferably K or R; most preferably K 84 A, T, D; P⁽⁵⁾, S, H, L, A, V, I, T, F, D, R,Y, N, Q, predominantly AG, E; preferably P 103 W W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, L, F, T, N, V, Q,P⁽⁶⁾, E, C; preferably W 104 G G, A, S, T, D, P, N, E, C, L; preferablyG 108 L, M or T; Q, L⁽⁷⁾, R, P, E, K, S, T, M, A, H; predominantly Lpreferably Q or L⁽⁷⁾ Notes: ⁽¹⁾In particular, but not exclusively, incombination with KERE or KQRE at positions 43-46. ⁽²⁾Usually as GLEW atpositions 44-47. ⁽³⁾Usually as KERE or KQRE at positions 43-46, e.g. asKEREL, KEREF, KQREL, KQREF, KEREG, KQREW or KQREG at positions 43-47.Alternatively, also sequences such as TERE (for example TEREL), TQRE(for example TQREL), KECE (for example KECEL or KECER), KQCE (forexample KQCEL), RERE (for example REREG), RQRE (for example RQREL, RQREFor RQREW), QERE (for example QEREG), QQRE, (for example QQREW, QQREL orQQREF), KGRE (for example KGREG), KDRE (for example KDREV) are possible.Some other possible, but less preferred sequences include for exampleDECKL and NVCEL. ⁽⁴⁾With both GLEW at positions 44-47 and KERE or KQREat positions 43-46. ⁽⁵⁾Often as KP or EP at positions 83-84 of naturallyoccurring V_(HH) domains. ⁽⁶⁾In particular, but not exclusively, incombination with GLEW at positions 44-47. ⁽⁷⁾With the proviso that whenpositions 44-47 are GLEW, position 108 is always Q in (non-humanized)V_(HH) sequences that also contain a W at 103. ⁽⁸⁾The GLEW group alsocontains GLEW-like sequences at positions 44-47, such as for exampleGVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER andELEW.

TABLE B-3 Some preferred but non-limiting combinations of HallmarkResidues in naturally occurring Nanobodies. For humanization of thesecombinations, reference is made to the specification. 11 37 44 45 47 8384 103 104 108 DP-47 (human) M V G L W R A W G L “KERE” group L F E R LK P W G Q L F E R F E P W G Q L F E R F K P W G Q L Y Q R L K P W G Q LF L R V K P Q G Q L F Q R L K P W G Q L F E R F K P W G Q “GLEW” group LV G L W K S W G Q M V G L W K P R G Q

In the Nanobodies, each amino acid residue at any other position thanthe Hallmark Residues can be any amino acid residue that naturallyoccurs at the corresponding position (according to the Kabat numbering)of a naturally occurring V_(HH) domain.

Such amino acid residues will be clear to the skilled person. Tables B-4to B-7 mention some non-limiting residues that can be present at eachposition (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4of naturally occurring V_(HH) domains. For each position, the amino acidresidue that most frequently occurs at each position of a naturallyoccurring V_(HH) domain (and which is the most preferred amino acidresidue for said position in a Nanobody) is indicated in bold; and otherpreferred amino acid residues for each position have been underlined(note: the number of amino acid residues that are found at positions26-30 of naturally occurring V_(HH) domains supports the hypothesisunderlying the numbering by Chothia (supra) that the residues at thesepositions already form part of CDR1).

In Tables B-4-B-7, some of the non-limiting residues that can be presentat each position of a human V_(H)3 domain have also been mentioned.Again, for each position, the amino acid residue that most frequentlyoccurs at each position of a naturally occurring human V_(H)3 domain isindicated in bold; and other preferred amino acid residues have beenunderlined.

For reference only, Tables B-4-B-7 also contain data on the V_(HH)entropy (“V_(HH) Ent.”) and V_(HH) variability (“V_(HH) Var.”) at eachamino acid position for a representative sample of 7732 V_(HH) sequences(including a.o. data kindly provided by David Lutje Hulsing and Prof.Theo Verrips of Utrecht University). The values for the V_(HH) entropyand the V_(HH) variability provide a measure for the variability anddegree of conservation of amino acid residues between the 7732 V_(HH)sequences analyzed: low values (i.e. <1, such as <0.5) indicate that anamino acid residue is highly conserved between the V_(HH) sequences(i.e. little variability). For example, the Gat position 9 and the W atposition 36 have values for the V_(HH) entropy of 0.01 and 0respectively, indicating that these residues are highly conserved andhave little variability (and in case of position 36 is W in all 7732sequences analysed), whereas for residues that form part of the CDR'sgenerally values of 1.5 or more are found (data not shown). Note thatthe data represented below support the hypothesis that the amino acidresidues at positions 27-30 and maybe even also at positions 93 and 94already form part of the CDR's (although the invention is not limited toany specific hypothesis or explanation, and as mentioned above, hereinthe numbering according to Kabat is used). For a general explanation ofsequence entropy, sequence variability and the methodology fordetermining the same, see Oliveira et al., PROTEINS: Structure, Functionand Genetics, 52: 544-552 (2003).

TABLE B-4 Non-limiting examples of amino acid residues in FR1 (for thefootnotes, see the footnotes to Table B-2) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 1 E, Q E, Q, K, D,A, G, R 0.47 5 2 V V, M, A, E, L 0.04 1 3 Q Q, K, P, H, F, R 0.04 1 4 LL, M, Q, P, R, F, V 0.02 1 5 V, L V, Q, M, E, A, L, P, K, R 0.35 3 6 EE, A, Q, D, K, H 0.21 5 7 S, T S, F, L, W, T 0.05 2 8 G, R G, R, E, V0.04 1 9 G G, R, V, A 0.01 1 10 G, V G, D, R, S, K, E, A, Q, N, T, V0.22 4 11 Hallmark residue: L, S, V, M, W, F, T, Q, E, A, 0.35 4 R, G,K, Y, N, P, I; preferably L 12 V, I V, A, L, M, E, G, T 0.11 2 13 Q, K,R Q, L, R, H, P, E, K, T, S, V, D, G, A, 0.46 3 N, M 14 P A, P, T, V, S,D, F, N, I, E, L, R, G, 0.92 5 Y, Q, H 15 G G, E 0 1 16 G, R G, D, E, A,S, N, V, R, K, T, P, C, L 0.47 4 17 S S, F, P, Y, T, A, C, R, N 0.14 218 L L, V, R, M, P, Q, S, A, T, K, H 0.06 1 19 R, K R, T, K, S, N, G, A,I, L, Q, F, E, V, 0.36 4 M 20 L L, F, V, I, P, H, S 0.18 3 21 S S, A, T,P, F, V, H, D, R, L, I, G 0.13 3 22 C C, W 0 1 23 A, T A, V, T, E, S, L,G, I, K, Q, R, D, F, 0.88 5 N, P, M 24 A A, D, V, T, H, Y, P, G, S, F,L, I, N, 0.78 9 Q, E, R 25 S S, P, T, A, F, L, N, Y, R, H, D, V, I, 0.22 W, G, K, Q, C 26 G G, E, R, V, T, A, S, K, D, L, I, Q, N, 0.45 6 F, Y,M, W, P, H 27 F R, F, S, P, L, G, I, N, T, D, H, V, E, 1.89 12 A, Y, K,M, Q, W, C 28 T T, I, S, A, P, F, D, N, V, R, M, L, G, 1.29 12 Y, K, E,H, W, Q 29 F, V F, L, S, V, I, A, W, Y, G, D, R, T, P, 1.23 11 N, E, M,H, Q, K, C 30 S, D, G S, D, N, G, R, T, A, E, I, Y, K, V, H, 1.55 12 L,F, W, M, P, C, Q

TABLE B-5 Non-limiting examples of amino acid residues in FR2 (for thefootnotes, see the footnotes to Table B-2) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 36 W W 0 1 37Hallmark residue: F⁽¹⁾, Y, V, L, A, H, S, I, W, C, 1.1 7 N, G, D, T, P,preferably F⁽¹⁾ or Y 38 R R, H, C, P, Y, L, V 0.01 1 39 Q Q, E, R, H, L,A, S, K, P, V, T, D 0.22 3 40 A A, V, T, P, G, S, D, I, L, R, N, F, Y,0.55 6 C, E, H 41 P, S, T P, S, A, L, T, Q, R, V, D, G, I, H 0.18 3 42 GG, E, A, R, D, V, W, T, Q, K, L, N, 0.1 2 H, M 43 K K, N, Q, E, R, T, L,S, M, D, G, A, 0.45 7 V, H, I, F, P 44 Hallmark residue: E⁽³⁾, Q⁽³⁾,G⁽²⁾, D, A, K, R, L, 1.11 4 P, S, V, H, T, N, W, M, I; preferably G⁽²⁾,E⁽³⁾. or Q⁽³⁾; most preferably G⁽²⁾ or Q⁽³⁾ 45 Hallmark residue: L⁽²⁾,R⁽³⁾, P, H, F, G, Q, S, E, 0.56 3 T, Y, C, I, D, V; preferably L⁽²⁾ orR⁽³⁾ 46 E, V E, D, A, Q, V, M, K, T, G, R, S, N, 0.42 4 I, L, F 47Hallmark residue: F⁽¹⁾, L⁽¹⁾ or W⁽²⁾ G, I, S, A, V, 1.64 11 M, R, Y, E,P, T, C, H, K, Q, N, D: preferably W⁽²⁾, L⁽¹⁾ or F⁽¹⁾ 48 V V, I, L, A,T, Q, F, M, G, E, R 0.35 5 49 S, A, G A, S, G, T, V, L, C, I, F, P, E,Y, M, 0.89 5 D, R

TABLE B-6 Non-limiting examples of amino acid residues in FR3 (for thefootnotes, see the footnotes to Table B-2) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 66 R R 0 1 67 F F,S, L, V, I, C, A, Y, M, G 0.1 1 68 T T, A, S, I, F, V, P, N, G, R, K, M,0.34 4 D, L, W, Q 69 I I, V, M, T, L, A, F, P, S, G, N 0.5 5 70 S S, T,A, F, P, V, Y, L, D, G, N, H, 0.22 4 W, E, C 71 R R, S, K, G, T, I, W,A, N, V, E, L, 0.61 7 M, F, D, Q, C 72 D, E D, N, E, G, V, A, H, L, S,T, I, Q, F, 0.34 4 P, Y, R 73 N, D, G N, D, S, K, I, Y, G, T, H, R, A,V, F, 0.65 9 L, E, M, P, C 74 A, S A, T, V, S, F, G, D, P, N, I, R, L,Y, 0.8 8 H, E, Q, K, W, M 75 K K, N, E, R, Q, A, G, T, M, S, L, D, 0.716 V, W, Y, I 76 N, S N, K, S, R, D, T, H, G, E, A, Y, I, 0.66 7 M, Q, L,W, P, F, V 77 S, T, I T, A, M, S, R, I, V, L, P, E, N, K, 0.72 7 G, W, Q78 L, A V, L, A, M, I, G, T, F, W, Q, S, E, N, 1.11 6 H 79 Y, H Y, F, D,S, H, N, T, A, L, W, V, C, 0.68 8 G, E, I, P, R 80 L L, M, V, P, F 0.052 81 Q Q, E, R, H, L, D, T, G, K, P, A, I, S, 0.38 4 N, Y, V, M 82 M M,I, L, V, A, T, S, K 0.12 3 82a N, G N, S, D, T, E, H, K, I, A, G, R, Y,L, 0.77 5 V, F, Q 82b S S, N, T, G, H, D, R, A, K, I, M, V, 0.72 8 F, E,P, Y, C, L 82c L L, V, M, P, A, T, G 0.08 2 83 Hallmark residue: R,K⁽⁵⁾, T, E⁽⁵⁾, Q, N, S, I, V, 0.66 6 G, M, L, A, D, Y, H; preferably Kor R; most preferably K 84 Hallmark residue: P⁽⁵⁾, S, H, L, A, V, I, T,F, D, 0.85 7 R, Y, N, Q, G, E; preferably P 85 E, G E, D, G, A, Q, V, S,N, K, T, R, L 0.27 3 86 D D, E, G, N 0.02 1 87 T, M T, S, A, M, R, P, K,E 0.15 3 88 A A, G, S, D, N, T, P, V 0.23 2 89 V, L V, I, L, E, A, R, T,D, F, M, N, S, K, 0.71 7 G, Q, H 90 Y Y, H, F, N 0 1 91 Y, H Y, F, R, S,H, T, I, V, L, N, D, C, Q, 0.6 7 W, A, E, M 92 C C, R, P 0 1 93 A, K, TA, N, T, K, G, V, R, Y, S, H, W, L, 1.33 10 F, Q, M, I, E, C, D 94 K, R,T A, K, V, T, R, L, G, S, D, Q, I, M, 1.55 12 F, Y, N, E, H, P, C, W

TABLE B-7 Non-limiting examples of amino acid residues in FR4 (for thefootnotes, see the footnotes to Table B-2) Amino acid residue(s): V_(HH)V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 103 Hallmarkresidue: W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, 0.54 6 L, F, T, N, V, Q, P⁽⁶⁾, E,C; preferably W 104 Hallmark residue: G, A, S, T, D, P, N, E, C, L;preferably G 0.13 3 105 Q, R Q, K, H, R, P, E, L, T, N, S, V, A, 0.52 5M, G 106 G G, R, E 0 1 107 T T, Q, I, A, S, N, R, V, D 0.24 3 108Hallmark residue: Q, L⁽⁷⁾, R, P, E, K, S, T, M, 0.3 4 A, H; preferably Qor L⁽⁷⁾ 109 V V, I, L 0 1 110 T T, S, N, A, I, F 0.01 1 111 V V, I, A0.01 1 112 S S, T, P, F, A 0.01 1 113 S S, T, A, L, P, F, E, V 0.04 1

Thus, in another preferred, but not limiting aspect, a Nanobody of theinvention can be defined as an amino acid sequence with the (general)structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) one or more of the amino acid residues at positions 11, 37, 44,    45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering    are chosen from the Hallmark residues mentioned in Table B-2;    and in which:-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In particular, a Nanobody of the invention can be an amino acid sequencewith the (general) structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) (preferably) one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    B-2 (it being understood that V_(HH) sequences will contain one or    more Hallmark residues; and that partially humanized Nanobodies will    usually, and preferably, [still] contain one or more Hallmark    residues [although it is also within the scope of the invention to    provide—where suitable in accordance with the invention—partially    humanized Nanobodies in which all Hallmark residues, but not one or    more of the other amino acid residues, have been humanized]; and    that in fully humanized Nanobodies, where suitable in accordance    with the invention, all amino acid residues at the positions of the    Hallmark residues will be amino acid residues that occur in a human    V_(H)3 sequence. As will be clear to the skilled person based on the    disclosure herein that such V_(HH) sequences, such partially    humanized Nanobodies with at least one Hallmark residue, such    partially humanized Nanobodies without Hallmark residues and such    fully humanized Nanobodies all form aspects of this invention);    and in which:-   ii) said amino acid sequence has at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 1 to    22, in which for the purposes of determining the degree of amino    acid identity, the amino acid residues that form the CDR sequences    (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are    disregarded;    and in which:-   iii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

TABLE B-8Representative amino acid sequences for Nanobodies of the KERE, GLEW and P, R, S 103 group.The CDR's are indicated with XXXX KERE sequence no. 1 SEQ ID NO: 1EVQLVESGGGLVQPGGSLRLSCAASGIPESXXXXXWFRQAPGKQRDSVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS KERE sequence no. 2SEQ ID NO: 2 QVKLEESGGGLVQAGGSLRLSCVGSGRTFSXXXXXWFRLAPGKEREFVAXXXXXRFTISRDTASNRGYLHMNNLTPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 3SEQ ID NO: 3 AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWFRQTPGREREFVAXXXXXRFTISRDNAKNMVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS KERE sequence no. 4SEQ ID NO: 4 QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTISRDDAKNTVWLHGSTLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 5SEQ ID NO: 5 AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIXXXXXWGQGTQVTVSS KERE sequence no. 6SEQ ID NO: 6 DVKFVESGGGLVQAGGSLRLSCVASGFNFDXXXXXWFRQAPGKEREEVAXXXXXRFTISSEKDKNSVYLQMNSLKPEDTALYICAGXXXXXWGRGTQVTVSS KERE sequence no. 7SEQ ID NO: 7 QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFTIARDSTKDTFCLQMNNLKPEDTAVYYCYAXXXXXWGQGTQVTVSS KERE sequence no. 8SEQ ID NO: 8 EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFTISTDNAKNTVHLLMNRVNAEDTALYYCAVXXXXXWGRGTRVTVSS KERE sequence no. 9SEQ ID NO: 9 QVQLVESGGGLVQPGGSLRLSCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTISGDNAKRAIYLQMNNLKPDDTAVYYCNRXXXXXWGQGTQVTVSP KERE sequence no. 10SEQ ID NO:10 QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGKEREFVAXXXXXRFTISRNATKNTLTLRMDSLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 11SEQ ID NO: 11 EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPGEKREFVAXXXXXRFTIARENAGNMVYLQMNNLKPDDTALYTCAAXXXXXWGRGTQVTVSS KERE sequence no. 12SEQ ID NO: 12 AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFTISRDSAKNMMYLQMNNLKPQDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 13SEQ ID NO: 13 AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPWQERDFVAXXXXXRFTISRDNYKDTVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 14SEQ ID NO: 14 AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGRDREFVAXXXXXRFTVSRDSAENTVALQMNSLKPEDTAVYYCAAXXXXXWGQGTRVTVSS KERE sequence no. 15SEQ ID NO: 15 QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTISRDYAGNTAFLQMDSLKPEDTGVYYCATXXXXXWGQGTQVTVSS KERE sequence no. 16SEQ ID NO: 16 EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPGKEREFVAXXXXXRFTVSRDNGKNTAYLRMNSLKPEDTADYYCAVXXXXXLGSGTQVTVSS GLEW sequence no. 1SEQ ID NO: 17 AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFTISRDNAKNTLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQVTVSS GLEW sequence no. 2SEQ ID NO: 18 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS GLEW sequence no. 3SEQ ID NO: 19 EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTISRDNAKNTLYLEMNSLSPEDTAMYYCGRXXXXXRSKGIQVTVSS P, R, S 103 sequence no. 1SEQ ID NO: 20 AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPGKEREFVAXXXXXRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAXXXXXRGQGTQVTVSS P, R, S 103 sequence no. 2SEQ ID NO: 21 DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRFTISRDNAKNMLYLHLNNLKSEDTAVYYCRRXXXXXLGQGTQVTVSS P, R, S 103 sequence no. 3SEQ ID NO: 22 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS

In particular, a Nanobody of the invention of the KERE group can be anamino acid sequence with the (general) structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which:

-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:-   ii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-9 Representative FW1 sequences for Nanobodies of the KERE-group.KERE FW1 sequence no. 1 SEQ ID NO: 23 QVQRVESGGGLVQAGGSLRLSCAASGRTSSKERE FW1 sequence no. 2 SEQ ID NO: 24 QVQLVESGGGLVQTGDSLSLSCSASGRTFSKERE FW1 sequence no. 3 SEQ ID NO: 25 QVKLEESGGGLVQAGDSLRLSCAATGRAFGKERE FW1 sequence no. 4 SEQ ID NO: 26 AVQLVESGGGLVQPGESLGLSCVASGRDFVKERE FW1 sequence no. 5 SEQ ID NO: 27 EVQLVESGGGLVQAGGSLRLSCEVLGRTAGKERE FW1 sequence no. 6 SEQ ID NO: 28 QVQLVESGGGWVQPGGSLRLSCAASETILSKERE FW1 sequence no. 7 SEQ ID NO: 29 QVQLVESGGGTVQPGGSLNLSCVASGNTFNKERE FW1 sequence no. 8 SEQ ID NO: 30 EVQLVESGGGLAQPGGSLQLSCSAPGFTLDKERE FW1 sequence no. 9 SEQ ID NO: 31 AQELEESGGGLVQAGGSLRLSCAASGRTFNand in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-10Representative FW2 sequences for Nanobodies of the KERE-group.KERE FW2 sequence no. 1 SEQ ID NO: 41 WFRQAPGKEREFVAKERE FW2 sequence no. 2 SEQ ID NO: 42 WFRQTPGREREFVAKERE FW2 sequence no. 3 SEQ ID NO: 43 WYRQAPGKQREMVAKERE FW2 sequence no. 4 SEQ ID NO: 44 WYRQGPGKQRELVAKERE FW2 sequence no. 5 SEQ ID NO: 45 WIRQAPGKEREGVSKERE FW2 sequence no. 6 SEQ ID NO: 46 WFREAPGKEREGISKERE FW2 sequence no. 7 SEQ ID NO: 47 WYRQAPGKERDLVAKERE FW2 sequence no. 8 SEQ ID NO: 48 WFROAPGKQREEVSKERE FW2 sequence no. 9 SEQ 1D NO: 49 WFRQPPGKVREFVGand in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-11Representative FW3 sequences for Nanobodies of the KERE-group.KERE FW3 sequence no. 1 SEQ ID NO: 50 RFTISRDNAKNTVYLQMNSLKPEDTAVYRCYFKERE FW3 sequence no. 2 SEQ ID NO: 51 RFAISRDNNKNTGYLQMNSLEPEDTAVYYCAAKERE FW3 sequence no. 3 SEQ ID NO: 52 RFTVARNNAKNTVNLEMNSLKPEDTAVYYCAAKERE FW3 sequence no. 4 SEQ ID NO: 53 RFTISRDIAKNTVDLLMNNLEPEDTAVYYCAAKERE FW3 sequence no. 5 SEQ ID NO: 54 RLTISRDNAVDTMYLQMNSLKPEDTAVYYCAAKERE FW3 sequence no. 6 SEQ ID NO: 55 RFTISRDNAKNTVYLQMDNVKPEDTAIYYCAAKERE FW3 sequence no. 7 SEQ ID NO: 56 RFTISKDSGKNTVYLQMTSLKPEDTAVYYCATKERE FW3 sequence no. 8 SEQ ID NO: 57 RFTISRDSAKNMMYLQMNNLKPODTAVYYCAAKERE FW3 sequence no. 9 SEQ ID NO: 58 RFTISRENDKSTVYLQLNSLKPEDTAVYYCAAKERE FW3 sequence no. 10 SEQ ID NO: 59 RFTISRDYAGNTAYLQMNSLKPEDTGVYYCATand in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-12 Representative FW4 sequences for Nanobodiesof the KERE-group. KERE FW4 sequence  SEQ ID NO: 60 WGQGTQVTVSS no. 1KERE FW4 sequence  SEQ ID NO: 61 WGKGTLVTVSS no. 2 KERE FW4 sequence SEQ ID NO: 62 RGQGTRVTVSS no. 3 KERE FW4 sequence  SEQ ID NO: 63WGLGTQVTISS no. 4and in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

Also, the above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

With regard to framework 1, it will be clear to the skilled person that,when an amino acid sequence as outlined above is generated by expressionof a nucleotide sequence, the first four amino acid sequences (i.e.amino acid residues 1-4 according to the Kabat numbering) may often bedetermined by the primer(s) that have been used to generate said nucleicacid. Thus, for determining the degree of amino acid identity, the firstfour amino acid residues are preferably disregarded.

Also, with regard to framework 1, and although amino acid positions 27to 30 are according to the Kabat numbering considered to be part of theframework regions (and not the CDR's), it has been found by analysis ofa database of more than 1000 V_(HH) sequences that the positions 27 to30 have a variability (expressed in terms of V_(HH) entropy and V_(HH)variability—see Tables B-4 to B-7) that is much greater than thevariability on positions 1 to 26. Because of this, for determining thedegree of amino acid identity, the amino acid residues at positions 27to 30 are preferably also disregarded.

In view of this, a Nanobody of the KERE class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) the amino acid residue at position 45 according to the Kabat    numbering is a charged amino acid (as defined herein) or a cysteine    residue, and position 44 is preferably an E;    and in which:-   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE B-13 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the KERE-group. KERE FW1 sequence no. 10 SEQ ID NO: 32VESGGGLVQPGGSLRLSCAASG KERE FW1 sequence no. 11 SEQ ID NO: 33VDSGGGLVQAGDSLKLSCALTG KERE FW1 sequence no. 12 SEQ ID NO: 34VDSGGGLVQAGDSLRLSCAASG KERE FW1 sequence no. 13 SEQ ID NO: 35VDSGGGLVEAGGSLRLSCQVSE KERE FW1 sequence no. 14 SEQ ID NO: 36QDSGGGSVQAGGSLKLSCAASG KERE FW1 sequence no. 15 SEQ ID NO: 37VQSGGRLVQAGDSLRLSCAASE KERE FW1 sequence no. 16 SEQ ID NO: 38VESGGTLVQSGDSLKLSCASST KERE FW1 sequence no. 17 SEQ ID NO: 39MESGGDSVQSGGSLTLSCVASG KERE FW1 sequence no. 18 SEQ ID NO: 40QASGGGLVQAGGSLRLSCSASVand in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of Nanobodies of the KERE-class;    and in which:-   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

A Nanobody of the GLEW class may be an amino acid sequence that iscomprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   i) preferably, when the Nanobody of the GLEW-class is a    non-humanized Nanobody, the amino acid residue in position 108 is Q;-   ii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-14Representative FW1 sequences for Nanobodies of the GLEW-group.GLEW FW1 sequence no. 1 SEQ ID NO: 64 QVQLVESGGGLVQPGGSLRLSCAASGFTFSGLEW FW1 sequence no. 2 SEQ ID NO: 65 EVHLVESGGGLVRPGGSLRLSCAAFGFIFKGLEW FW1 sequence no. 3 SEQ ID NO: 66 QVKLEESGGGLAQPGGSLRLSCVASGFTFSGLEW FW1 sequence no. 4 SEQ ID NO: 67 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTGLEW FW1 sequence no. 5 SEQ ID NO: 68 EVQLVESGGGLALPGGSLTLSCVFSGSTFSand in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-15 Representative FW2 sequences for Nanobodiesof the GLEW-group. GLEW FW2 sequence  SEQ ID NO: 72 WVRQAPGKVLEWVS no. 1GLEW FW2 sequence  SEQ ID NO: 73 WVRRPPGKGLEWVS no. 2 GLEW FW2 sequence SEQ ID NO: 74 WVRQAPGMGLEWVS no. 3 GLEW FW2 sequence  SEQ ID NO: 75WVRQAPGKEPEWVS no. 4 GLEW FW2 sequence  SEQ ID NO: 76 WVRQAPGKDQEWVSno. 5 GLEW FW2 sequence  SEQ ID NO: 77 WVRQAPGKAEEWVS no. 6GLEW FW2 sequence  SEQ ID NO: 78 WVRQAPGKGLEWVA no. 7 GLEW FW2 sequence SEQ ID NO: 79 WVRQAPGRATEWVS no. 8and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-16Representative FW3 sequences for Nanobodies of the GLEW-group.GLEW FW3 sequence no. 1 SEQ ID NO: 80 RFTISRDNAKNTLYLQMNSLKPEDTAVYYCVKGLEW FW3 sequence no. 2 SEQ ID NO: 81 RFTISRDNARNTLYLQMDSLIPEDTALYYCARGLEW FW3 sequence no. 3 SEQ ID NO: 82 RFTSSRDNAKSTLYLQMNDLKPEDTALYYCARGLEW FW3 sequence no. 4 SEQ ID NO: 83 RFIISRDNAKNTLYLQMNSLGPEDTAMYYCQRGLEW FW3 sequence no. 5 SEQ ID NO: 84 RFTASRDNAKNTLYLQMNSLKSEDTARYYCARGLEW FW3 sequence no. 6 SEQ ID NO: 85 RFTISRDNAKNTLYLQMDDLQSEDTAMYYCGRand in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-17 Representative FW4 sequences for Nanobodiesof the GLEW-group. GLEW FW4 sequence  SEQ ID NO: 86 GSQGTQVTVSS no. 1GLEW FW4 sequence  SEQ ID NO: 87 LRGGTQVTVSS no. 2 GLEW FW4 sequence SEQ ID NO: 88 RGQGTLVTVSS no. 3 GLEW FW4 sequence  SEQ ID NO: 89RSRGIQVTVSS no. 4 GLEW FW4 sequence  SEQ ID NO: 90 WGKGTQVTVSS no. 5GLEW FW4 sequence  SEQ ID NO: 91 WGQGTQVTVSS no. 6and in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the GLEW class may be an amino acidsequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) preferably, when the Nanobody of the GLEW-class is a    non-humanized Nanobody, the amino acid residue in position 108 is Q;    and in which:-   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE B-18 Representative FW1 sequences (amino acid residues5 to 26) for Nanobodies of the KERE-group. GLEW FW1 sequence no. 6SEQ ID NO: 69 VESGGGLVQPGGSLRLSCAASG GLEW FW1 sequence no. 7SEQ ID NO: 70 EESGGGLAQPGGSLRLSCVASG GLEW FWI sequence no. 8SEQ ID NO: 71 VESGGGLALPGGSLTLSCVFSGand in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4    of Nanobodies of the GLEW-class;    and in which:-   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein. Inthe above Nanobodies, one or more of the further Hallmark residues arepreferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

A Nanobody of the P, R, S 103 class may be an amino acid sequence thatis comprised of four framework regions/sequences interrupted by threecomplementarity determining regions/sequences, in which

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or S, and more preferably R;    and in which:-   iii) FR1 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-19Representative FW1 sequences for Nanobodies of the P, R, S 103-group.P, R, S 103 FW1 sequence no. 1 SEQ ID NO: 92AVQLVESGGGLVQAGGSLRLSCAASGRTFS P, R, S 103 FW1 sequence no. 2SEQ ID NO: 93 QVQLQESGGGMVQPGGSLRLSCAASGFDFGP, R, S 103 FW1 sequence no. 3 SEQ ID N0: 94EVHLVESGGGLVRPGGSLRLSCAAFGFIFK P, R, S 103 FW1 sequence no. 4SEQ ID NO: 95 QVQLAESGGGLVQPGGSLKLSCAASRTIVSP, R, S 103 FW1 sequence no. 5 SEQ ID NO: 96QEHLVESGGGLVDIGGSLRLSCAASERIFS P, R, S 103 FW1 sequence no. 6SEQ ID NO: 97 QVKLEESGGGLAQPGGSLRLSCVASGFTFSP, R, S 103 FW1 sequence no. 7 SEQ ID NO: 98EVQLVESGGGLVQPGGSLRLSCVCVSSGCT P, R, S 103 FW1 sequence no. 8SEQ ID NO: 99 EVQLVESGGGLALPGGSLTLSCVFSGSTFSand in which

-   iv) FR2 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-20 Representative FW2 sequences for Nanobodies of theP, R, S 103-group. P, R, S 103 FW2 sequence no. 1 SEQ ID NO: 102WFRQAPGKEREFVA P, R, S 103 FW2 sequence no. 2 SEQ ID NO: 103WVRQAPGKVLEWVS P, R, S 103 FW2 sequence no. 3 SEQ ID NO: 104WVRRPPGKGLEWVS P, R, S 103 FW2 sequence no. 4 SEQ ID NO: 105WIRQAPGKEREGVS P, R, S 103 FW2 sequence no. 5 SEQ ID NO: 106WVRQYPGKEPEWVS P, R, S 103 FW2 sequence no. 6 SEQ ID NO: 107WFRQPPGKEHEFVA P, R, S 103 FW2 sequence no. 7 SEQ ID NO: 108WYRQAPGKRTELVA P, R, S 103 FW2 sequence no. 8 SEQ ID NO: 109WLRQAPGQGLEWVS P, R, S 103 FW2 sequence no. 9 SEQ ID NO: 110WLRQTPGKGLEWVG P, R, S 103 FW2 sequence no. 10 SEQ ID NO: 111WVRQAPGKAEEFVSand in which:

-   v) FR3 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-21Representative FW3 sequences for Nanobodies of the P, R, S 103-group.P, R, S 103 FW3 sequence no. 1 SEQ ID NO: 112RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA P, R, S 103 FW3 sequence no. 2SEQ ID NO: 113 RFTISRDNARNTLYLQMDSLIPEDTALYYCARP, R, S 103 FW3 sequence no. 3 SEQ ID NO: 114RFTISRDNAKNEMYLQMNNLKTEDTGVYWCGA P, R, S 103 FW3 sequence no. 4SEQ ID NO: 115 RFTISSDSNRNMIYLQMNNLKPEDTAVYYCAAP, R, S 103 FW3 sequence no. 5 SEQ ID NO: 116RFTISRDNAKNMLYLHLNNLKSEDTAVYYCRR P, R, S 103 FW3 sequence no. 6SEQ ID NO: 117 RFTISRDNAKKTVYLRLNSLNPEDTAVYSCNLP, R, S 103 FW3 sequence no. 7 SEQ ID NO: 118RFKISRDNAKKTLYLQMNSLGPEDTAMYYCQR P, R, S 103 FW3 sequence no. 8SEQ ID NO: 119 RFTVSRDNGKNTAYLRMNSLKPEDTADYYCAVand in which:

-   vi) FR4 is an amino acid sequence that has at least 80% amino acid    identity with at least one of the following amino acid sequences:

TABLE B-22 Representative FW4 sequences for Nanobodiesof the P, R, S 103-group. P, R, S 103 FW4 sequence no. 1 SEQ ID NO: 120RGQGTQVTVSS P, R, S 103 FW4 sequence no. 2 SEQ ID NO: 121 LRGGTQVTVSSP, R, S 103 FW4 sequence no. 3 SEQ ID NO: 122 GNKGTLVTVSSP, R, S 103 FW4 sequence no. 4 SEQ ID NO: 123 SSPGTQVTVSSP, R, S 103 FW4 sequence no. 5 SEQ ID NO: 124 SSQGTLVTVSSP, R, S 103 FW4 sequence no. 6 SEQ ID NO: 125 RSRGIQVTVSSand in which:

-   vii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably    as defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled personthat, for determining the degree of amino acid identity, the amino acidresidues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the P, R, S 103 class may be an aminoacid sequence that is comprised of four framework regions/sequencesinterrupted by three complementarity determining regions/sequences, inwhich:

-   i) the amino acid residue at position 103 according to the Kabat    numbering is different from W;    and in which:-   ii) preferably the amino acid residue at position 103 according to    the Kabat numbering is P, R or S, and more preferably R;    and in which:-   iii) FR1 is an amino acid sequence that, on positions 5 to 26 of the    Kabat numbering, has at least 80% amino acid identity with at least    one of the following amino acid sequences:

TABLE B-23 Representative FW1 sequences (amino acid residues 5 to 26)for Nanobodies of the P, R, S 103-group. P, R, S 103 FW1 sequence no. 9SEQ ID NO: 100 VESGGGLVQAGGSLRLSCAASG P, R, S 103 FW1 sequence no. 10SEQ ID NO: 101 AESGGGLVQPGGSLKLSCAASRand in which:

-   iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of    Nanobodies of the P, R, S 103 class;    and in which:-   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as    defined according to one of the preferred aspects herein, and are    more preferably as defined according to one of the more preferred    aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may behumanized Nanobodies. When the above Nanobody sequences are V_(HH)sequences, they may be suitably humanized, as further described herein.When the Nanobodies are partially humanized Nanobodies, they mayoptionally be further suitably humanized, again as described herein.

In the above Nanobodies, one or more of the further Hallmark residuesare preferably as described herein (for example, when they are V_(HH)sequences or partially humanized Nanobodies).

In another preferred, but non-limiting aspect, the invention relates toa Nanobody as described above, in which the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 308-333 (see Table A-1). This degree of amino acid identity canfor example be determined by determining the degree of amino acididentity (in a manner described herein) between said Nanobody and one ormore of the sequences of SEQ ID NO's: 308-333 (see Table A-1), in whichthe amino acid residues that form the framework regions are disregarded.Such Nanobodies can be as further described herein.

As already mentioned herein, another preferred but non-limiting aspectof the invention relates to a Nanobody with an amino acid sequence thatis chosen from the group consisting of SEQ ID NO's: 308-333 (see TableA-1) or from the group consisting of from amino acid sequences that havemore than 80%, preferably more than 90%, more preferably more than 95%,such as 99% or more sequence identity (as defined herein) with at leastone of the amino acid sequences of SEQ ID NO's: 308-333 (see Table A-1).

Also, in the above Nanobodies:

-   i) any amino acid substitution (when it is not a humanizing    substitution as defined herein) is preferably, and compared to the    corresponding amino acid sequence of SEQ ID NO's: 308-333 (see Table    A-1), a conservative amino acid substitution, (as defined herein);    and/or:-   ii) its amino acid sequence preferably contains either only amino    acid substitutions, or otherwise preferably no more than 5,    preferably no more than 3, and more preferably only 1 or 2 amino    acid deletions or insertions, compared to the corresponding amino    acid sequence of SEQ ID NO's: 308-333 (see Table A-1);    and/or-   iii) the CDR's may be CDR's that are derived by means of affinity    maturation, for example starting from the CDR's of to the    corresponding amino acid sequence of SEQ ID NO's: 308-333 (see Table    A-1).

Preferably, the CDR sequences and FR sequences in the Nanobodies of theinvention are such that the Nanobodies of the invention (andpolypeptides of the invention comprising the same):

-   -   bind to multiscavenger receptors with a dissociation constant        (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably        10⁻⁷ to 10⁻¹² moles/liter or less and more preferably 10⁻⁸ to        10⁻¹² moles/liter (i.e. with an association constant (K_(A)) of        10⁵ to 10¹² liter/moles or more, and preferably 10⁷ to 10¹²        liter/moles or more and more preferably 10⁸ to 10¹²        liter/moles);        and/or such that they:    -   bind to multiscavenger receptors with a k_(on)-rate of between        10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹        and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷        M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to multiscavenger receptors with a k_(off) rate between 1        s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t₁₁₂ of multiple days), preferably between 10⁻²        s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹,        such as between 10⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, CDR sequences and FR sequences present in the Nanobodies ofthe invention are such that the Nanobodies of the invention will bind tomultiscavenger receptors with an affinity less than 500 nM, preferablyless than 200 nM, more preferably less than 10 nM, such as less than 500pM.

According to one non-limiting aspect of the invention, a Nanobody may beas defined herein, but with the proviso that it has at least “one aminoacid difference” (as defined herein) in at least one of the frameworkregions compared to the corresponding framework region of a naturallyoccurring human V_(H) domain, and in particular compared to thecorresponding framework region of DP-47. More specifically, according toone non-limiting aspect of the invention, a Nanobody may be as definedherein, but with the proviso that it has at least “one amino aciddifference” (as defined herein) at least one of the Hallmark residues(including those at positions 108, 103 and/or 45) compared to thecorresponding framework region of a naturally occurring human V_(H)domain, and in particular compared to the corresponding framework regionof DP-47. Usually, a Nanobody will have at least one such amino aciddifference with a naturally occurring V_(H) domain in at least one ofFR2 and/or FR4, and in particular at least one of the Hallmark residuesin FR2 and/or FR4 (again, including those at positions 108, 103 and/or45).

Also, a humanized Nanobody of the invention may be as defined herein,but with the proviso that it has at least “one amino acid difference”(as defined herein) in at least one of the framework regions compared tothe corresponding framework region of a naturally occurring V_(HH)domain. More specifically, according to one non-limiting aspect of theinvention, a humanized. Nanobody may be as defined herein, but with theproviso that it has at least “one amino acid difference” (as definedherein) at least one of the Hallmark residues (including those atpositions 108, 103 and/or 45) compared to the corresponding frameworkregion of a naturally occurring V_(HH) domain. Usually, a humanizedNanobody will have at least one such amino acid difference with anaturally occurring V_(HH) domain in at least one of FR2 and/or FR4, andin particular at least one of the Hallmark residues in FR2 and/or FR4(again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scopeof the invention to use natural or synthetic analogs, mutants, variants,alleles, homologs and orthologs (herein collectively referred to as“analogs”) of the Nanobodies of the invention as defined herein, and inparticular analogs of the Nanobodies of SEQ ID NO's 308-333 (see TableA-1). Thus, according to one aspect of the invention, the term “Nanobodyof the invention” in its broadest sense also covers such analogs.

Generally, in such analogs, one or more amino acid residues may havebeen replaced, deleted and/or added, compared to the Nanobodies of theinvention as defined herein. Such substitutions, insertions or deletionsmay be made in one or more of the framework regions and/or in one ormore of the CDR's. When such substitutions, insertions or deletions aremade in one or more of the framework regions, they may be made at one ormore of the Hallmark residues and/or at one or more of the otherpositions in the framework residues, although substitutions, insertionsor deletions at the Hallmark residues are generally less preferred(unless these are suitable humanizing substitutions as describedherein).

By means of non-limiting examples, a substitution may for example be aconservative substitution (as described herein) and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another V_(HH) domain (see Tables B-4 toB-7 for some non-limiting examples of such substitutions), although theinvention is generally not limited thereto. Thus, any one or moresubstitutions, deletions or insertions, or any combination thereof, thateither improve the properties of the Nanobody of the invention or thatat least do not detract too much from the desired properties or from thebalance or combination of desired properties of the Nanobody of theinvention (i.e. to the extent that the Nanobody is no longer suited forits intended use) are included within the scope of the invention. Askilled person will generally be able to determine and select suitablesubstitutions, deletions or insertions, or suitable combinations ofthereof, based on the disclosure herein and optionally after a limiteddegree of routine experimentation, which may for example involveintroducing a limited number of possible substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

For example, and depending on the host organism used to express theNanobody or polypeptide of the invention, such deletions and/orsubstitutions may be designed in such a way that one or more sites forpost-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art. Alternatively, substitutions or insertions may be designedso as to introduce one or more sites for attachment of functional groups(as described herein), for example to allow site-specific pegylation(again as described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH)variability given in Tables B-4 to B-7 above, some amino acid residuesin the framework regions are more conserved than others. Generally,although the invention in its broadest sense is not limited thereto, anysubstitutions, deletions or insertions are preferably made at positionsthat are less conserved. Also, generally, amino acid substitutions arepreferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to multiscavengerreceptors with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein for the Nanobodiesof the invention.

The analogs are preferably also such that they retain the favourableproperties the Nanobodies, as described herein.

Also, according to one preferred aspect, the analogs have a degree ofsequence identity of at least 70%, preferably at least 80%, morepreferably at least 90%, such as at least 95% or 99% or more; and/orpreferably have at most 20, preferably at most 10, even more preferablyat most 5, such as 4, 3, 2 or only 1 amino acid difference (as definedherein), with one of the Nanobodies of SEQ ID NOs: 308-333 (see TableA-1).

Also, the framework sequences and CDR's of the analogs are preferablysuch that they are in accordance with the preferred aspects definedherein. More generally, as described herein, the analogs will have (a) aQ at position 108; and/or (b) a charged amino acid or a cysteine residueat position 45 and preferably an E at position 44, and more preferably Eat position 44 and R at position 45; and/or (c) P, R or S at position103.

One preferred class of analogs of the Nanobodies of the inventioncomprise Nanobodies that have been humanized (i.e. compared to thesequence of a naturally occurring Nanobody of the invention). Asmentioned in the background art cited herein, such humanizationgenerally involves replacing one or more amino acid residues in thesequence of a naturally occurring V_(HH) with the amino acid residuesthat occur at the same position in a human V_(H) domain, such as a humanV_(H)3 domain. Examples of possible humanizing substitutions orcombinations of humanizing substitutions will be clear to the skilledperson, for example from the Tables herein, from the possible humanizingsubstitutions mentioned in the background art cited herein, and/or froma comparision between the sequence of a Nanobody and the sequence of anaturally occurring human V_(H) domain.

The humanizing substitutions should be chosen such that the resultinghumanized Nanobodies still retain the favourable properties ofNanobodies as defined herein, and more preferably such that they are asdescribed for analogs in the preceding paragraphs. A skilled person willgenerally be able to determine and select suitable humanizingsubstitutions or suitable combinations of humanizing substitutions,based on the disclosure herein and optionally after a limited degree ofroutine experimentation, which may for example involve introducing alimited number of possible humanizing substitutions and determiningtheir influence on the properties of the Nanobodies thus obtained.

Generally, as a result of humanization, the Nanobodies of the inventionmay become more “human-like”, while still retaining the favorableproperties of the Nanobodies of the invention as described herein. As aresult, such humanized Nanobodies may have several advantages, such as areduced immunogenicity, compared to the corresponding naturallyoccurring V_(HH) domains. Again, based on the disclosure herein andoptionally after a limited degree of routine experimentation, theskilled person will be able to select humanizing substitutions orsuitable combinations of humanizing substitutions which optimize orachieve a desired or suitable balance between the favourable propertiesprovided by the humanizing substitutions on the one hand and thefavourable properties of naturally occurring V_(HH) domains on the otherhand.

The Nanobodies of the invention may be suitably humanized at anyframework residue(s), such as at one or more Hallmark residues (asdefined herein) or at one or more other framework residues (i.e.non-Hallmark residues) or any suitable combination thereof. Onepreferred humanizing substitution for Nanobodies of the “P, R, S-103group” or the “KERE group” is Q108 into L108. Nanobodies of the “GLEWclass” may also be humanized by a Q108 into L108 substitution, providedat least one of the other Hallmark residues contains a carnelid(camelizing) substitution (as defined herein). For example, as mentionedabove, one particularly preferred class of humanized Nanobodies has GLEWor a GLEW-like sequence at positions 44-47; P, R or S (and in particularR) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding thesame, can be provided in any manner known per se, for example using oneor more of the techniques mentioned on pages 103 and 104 of WO08/020,079.

As mentioned there, it will be also be clear to the skilled person thatthe Nanobodies of the invention (including their analogs) can bedesigned and/or prepared starting from human V_(H) sequences (i.e. aminoacid sequences or the corresponding nucleotide sequences), such as forexample from human V_(H)3 sequences such as DP-47, DP-51 or DP-29, i.e.by introducing one or more camelizing substitutions (i.e. changing oneor more amino acid residues in the amino acid sequence of said humanV_(H) domain into the amino acid residues that occur at thecorresponding position in a V_(HH) domain), so as to provide thesequence of a Nanobody of the invention and/or so as to confer thefavourable properties of a Nanobody to the sequence thus obtained.Again, this can generally be performed using the various methods andtechniques referred to in the previous paragraph, using an amino acidsequence and/or nucleotide sequence for a human. V_(H) domain as astarting point.

Some preferred, but non-limiting camelizing substitutions can be derivedfrom Tables B-4-B-7. It will also be clear that camelizing substitutionsat one or more of the Hallmark residues will generally have a greaterinfluence on the desired properties than substitutions at one or more ofthe other amino acid positions, although both and any suitablecombination thereof are included within the scope of the invention. Forexample, it is possible to introduce one or more camelizingsubstitutions that already confer at least some the desired properties,and then to introduce further camelizing substitutions that eitherfurther improve said properties and/or confer additional favourableproperties. Again, the skilled person will generally be able todetermine and select suitable camelizing substitutions or suitablecombinations of camelizing substitutions, based on the disclosure hereinand optionally after a limited degree of routine experimentation, whichmay for example involve introducing a limited number of possiblecamelizing substitutions and determining whether the favourableproperties of Nanobodies are obtained or improved (i.e. compared to theoriginal V_(H) domain). Generally, however, such camelizingsubstitutions are preferably such that the resulting an amino acidsequence at least contains (a) a Q at position 108; and/or (b) a chargedamino acid or a cysteine residue at position 45 and preferably also an Eat position 44, and more preferably E at position 44 and R at position45; and/or (c) P, R or S at position 103; and optionally one or morefurther camelizing substitutions. More preferably, the camelizingsubstitutions are such that they result in a Nanobody of the inventionand/or in an analog thereof (as defined herein), such as in a humanizedanalog and/or preferably in an analog that is as defined in thepreceding paragraphs.

Nanobodies can also be derived from V_(H) domains by the incorporationof substitutions that are rare in nature, but nonetheless, structurallycompatible with the VH domain fold. For example, but without beinglimiting, these substitutions may include on or more of the following:Gly at position 35, Ser, Val or Thr at position 37, Ser, Thr, Arg, Lys,H is, Asp or Glu at position 39, Glu or H is at position 45, Trp, Leu,Val, Ala, Thr, or Glu at position 47, S or R at position 50. (Barthelemyet al. J Biol. Chem. 2008 Feb. 8; 283(6):3639-54. Epub 2007 Nov. 28)

As will also be clear from the disclosure herein, it is also within thescope of the invention to use parts or fragments, or combinations of twoor more parts or fragments, of the Nanobodies of the invention asdefined herein, and in particular parts or fragments of the Nanobodiesof SEQ ID NO's: 308-333 (see Table A-1). Thus, according to one aspectof the invention, the term “Nanobody of the invention” in its broadestsense also covers such parts or fragments.

Generally, such parts or fragments of the Nanobodies of the invention(including analogs thereof) have amino acid sequences in which, comparedto the amino acid sequence of the corresponding full length Nanobody ofthe invention (or analog thereof), one or more of the amino acidresidues at the N-terminal end, one or more amino acid residues at theC-terminal end, one or more contiguous internal amino acid residues, orany combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind tomultiscavenger receptors with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined hereinfor the Nanobodies of the invention.

Any part or fragment is preferably such that it comprises at least 10contiguous amino acid residues, preferably at least 20 contiguous aminoacid residues, more preferably at least 30 contiguous amino acidresidues, such as at least 40 contiguous amino acid residues, of theamino acid sequence of the corresponding full length Nanobody of theinvention.

Also, any part or fragment is such preferably that it comprises at leastone of CDR1, CDR2 and/or CDR3 or at least part thereof (and inparticular at least CDR3 or at least part thereof). More preferably, anypart or fragment is such that it comprises at least one of the CDR's(and preferably at least CDR3 or part thereof) and at least one otherCDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connectedby suitable framework sequence(s) or at least part thereof. Morepreferably, any part or fragment is such that it comprises at least oneof the CDR's (and preferably at least CDR3 or part thereof) and at leastpart of the two remaining CDR's, again preferably connected by suitableframework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect,such a part or fragment comprises at least CDR3, such as FR3, CDR3 andFR4 of the corresponding full length Nanobody of the invention, i.e. asfor example described in the International application WO 03/050531(Lasters et al.).

As already mentioned above, it is also possible to combine two or moreof such parts or fragments (i.e. from the same or different Nanobodiesof the invention), i.e. to provide an analog (as defined herein) and/orto provide further parts or fragments (as defined herein) of a Nanobodyof the invention. It is for example also possible to combine one or moreparts or fragments of a Nanobody of the invention with one or more partsor fragments of a human V_(II) domain.

According to one preferred aspect, the parts or fragments have a degreeof sequence identity of at least 50%, preferably at least 60%, morepreferably at least 70%, even more preferably at least 80%, such as atleast 90%, 95% or 99% or more with one of the Nanobodies of SEQ ID NOs308-333 (see Table A-1).

The parts and fragments, and nucleic acid sequences encoding the same,can be provided and optionally combined in any manner known per se. Forexample, such parts or fragments can be obtained by inserting a stopcodon in a nucleic acid that encodes a full-sized Nanobody of theinvention, and then expressing the nucleic acid thus obtained in amanner known per se (e.g. as described herein). Alternatively, nucleicacids encoding such parts or fragments can be obtained by suitablyrestricting a nucleic acid that encodes a full-sized Nanobody of theinvention or by synthesizing such a nucleic acid in a manner known perse. Parts or fragments may also be provided using techniques for peptidesynthesis known per se.

The invention in its broadest sense also comprises derivatives of theNanobodies of the invention. Such derivatives can generally be obtainedby modification, and in particular by chemical and/or biological (e.genzymatical) modification, of the Nanobodies of the invention and/or ofone or more of the amino acid residues that form the Nanobodies of theinvention.

Examples of such modifications, as well as examples of amino acidresidues within the Nanobody sequence that can be modified in such amanner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the Nanobody of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the Nanobody of the invention. Example of suchfunctional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that increase the half-life, the solubility and/or theabsorption of the Nanobody of the invention, that reduce theimmunogenicity and/or the toxicity of the Nanobody of the invention,that eliminate or attenuate any undesirable side effects of the Nanobodyof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the Nanobodies and/orpolypeptides of the invention; or any combination of two or more of theforegoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a Nanobody of theinvention, a Nanobody of the invention may be modified so as to suitablyintroduce one or more cysteine residues for attachment of PEG, or anamino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a Nanobodyof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG isused with a molecular weight of more than 5000, such as more than 10,000and less than 200,000, such as less than 100,000; for example in therange of 20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled Nanobody. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and for example include, but are not limited to, thefluorescent labels, phosphorescent labels, chemiluminescent labels,bioluminescent labels, radio-isotopes, metals, metal chelates, metalliccations, chromophores and enzymes, such as those mentioned on page 109of WO 08/020,079. Other suitable labels will be clear to the skilledperson, and for example include moieties that can be detected using NMRor ESR spectroscopy.

Such labelled Nanobodies and polypeptides of the invention may forexample be used for in vitro, in vivo or in situ assays (includingimmunoassays known per se such as ELISA, RIA, ETA and other “sandwichassays”, etc.) as well as in vivo diagnostic and imaging purposes,depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethylenetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the Nanobody of the invention to another protein, polypeptide orchemical compound that is bound to the other half of the binding pair,i.e. through formation of the binding pair. For example, a Nanobody ofthe invention may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated Nanobody may be used as areporter, for example in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may for example also be used to bind the Nanobody of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the Nanobody of the invention.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell, the Nanobodies of the invention may also be linked to a toxin orto a toxic residue or moiety. Examples of toxic moieties, compounds orresidues which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic compound will be clear to the skilledperson and can for example be found in the prior art cited above and/orin the further description herein. One example is the so-called ADEPT™technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw, Biotechnol.Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to multiscavengerreceptors with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein for the Nanobodiesof the invention.

As mentioned above, the invention also relates to proteins orpolypeptides that essentially consist of or comprise at least oneNanobody of the invention. By “essentially consist of” is meant that theamino acid sequence of the polypeptide of the invention either isexactly the same as the amino acid sequence of a Nanobody of theinvention or corresponds to the amino acid sequence of a Nanobody of theinvention which has a limited number of amino acid residues, such as1-20 amino acid residues, for example 1-10 amino acid residues andpreferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 aminoacid residues, added at the amino terminal end, at the carboxy terminalend, or at both the amino terminal end and the carboxy terminal end ofthe amino acid sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the Nanobody and may or may notadd further functionality to the Nanobody. For example, such amino acidresidues:

-   -   can comprise an N-terminal Met residue, for example as result of        expression in a heterologous host cell or host organism.    -   may form a signal sequence or leader sequence that directs        secretion of the Nanobody from a host cell upon synthesis.        Suitable secretory leader peptides will be clear to the skilled        person, and may be as further described herein. Usually, such a        leader sequence will be linked to the N-terminus of the        Nanobody, although the invention in its broadest sense is not        limited thereto;    -   may form a sequence or signal that allows the Nanobody to be        directed towards and/or to penetrate or enter into specific        organs, tissues, cells, or parts or compartments of cells,        and/or that allows the Nanobody to penetrate or cross a        biological barrier such as a cell membrane, a cell layer such as        a layer of epithelial cells, a tumor including solid tumors, or        the blood-brain-barrier. Examples of such amino acid sequences        will be clear to the skilled person and include those mentioned        in paragraph c) on page 112 of WO 08/020,079.    -   may form a “tag”, for example an amino acid sequence or residue        that allows or facilitates the purification of the Nanobody, for        example using affinity techniques directed against said sequence        or residue. Thereafter, said sequence or residue may be removed        (e.g. by chemical or enzymatical cleavage) to provide the        Nanobody sequence (for this purpose, the tag may optionally be        linked to the Nanobody sequence via a cleavable linker sequence        or contain a cleavable motif). Some preferred, but non-limiting        examples of such residues are multiple histidine residues,        glutatione residues and a myc-tag (see for example SEQ ID NO:31        of WO 06/12282).    -   may be one or more amino acid residues that have been        functionalized and/or that can serve as a site for attachment of        functional groups. Suitable amino acid residues and functional        groups will be clear to the skilled person and include, but are        not limited to, the amino acid residues and functional groups        mentioned herein for the derivatives of the Nanobodies of the        invention.

According to another aspect, a polypeptide of the invention comprises aNanobody of the invention, which is fused at its amino terminal end, atits carboxy terminal end, or both at its amino terminal end and at itscarboxy terminal end to at least one further amino acid sequence, i.e.so as to provide a fusion protein comprising said Nanobody of theinvention and the one or more further amino acid sequences. Such afusion will also be referred to herein as a “Nanobody fusion”.

The one or more further amino acid sequence may be any suitable and/ordesired amino acid sequences. The further amino acid sequences may ormay not change, alter or otherwise influence the (biological) propertiesof the Nanobody, and may or may not add further functionality to theNanobody or the polypeptide of the invention. Preferably, the furtheramino acid sequence is such that it confers one or more desiredproperties or functionalities to the Nanobody or the polypeptide of theinvention.

For example, the further amino acid sequence may also provide a secondbinding site, which binding site may be directed against any desiredprotein, polypeptide, antigen, antigenic determinant or epitope(including but not limited to the same protein, polypeptide, antigen,antigenic determinant or epitope against which the Nanobody of theinvention is directed, or a different protein, polypeptide, antigen,antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilledperson, and may generally comprise all amino acid sequences that areused in peptide fusions based on conventional antibodies and fragmentsthereof (including but not limited to ScFv's and single domainantibodies). Reference is for example made to the review by Holliger andHudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, such an amino acid sequence may be an amino acid sequencethat increases the half-life, the solubility, or the absorption, reducesthe immunogenicity or the toxicity, eliminates or attenuates undesirableside effects, and/or confers other advantageous properties to and/orreduces the undesired properties of the polypeptides of the invention,compared to the Nanobody of the invention per se. Some non-limitingexamples of such amino acid sequences are serum proteins, such as humanserum albumin (see for example WO 00/27435) or haptenic molecules (forexample haptens that are recognized by circulating antibodies, see forexample WO 98/22141).

In particular, it has been described in the art that linking fragmentsof immunoglobulins (such as V_(H) domains) to serum albumin or tofragments thereof can be used to increase the half-life. Reference isfor made to WO 00/27435 and WO 01/077137). According to the invention,the Nanobody of the invention is preferably either directly linked toserum albumin (or to a suitable fragment thereof) or via a suitablelinker, and in particular via a suitable peptide linked so that thepolypeptide of the invention can be expressed as a genetic fusion(protein). According to one specific aspect, the Nanobody of theinvention may be linked to a fragment of serum albumin that at leastcomprises the domain III of serum albumin or part thereof. Reference isfor example made to WO 07/112,940 of Ablynx N.V.

Alternatively, the further amino acid sequence may provide a secondbinding site or binding unit that is directed against a serum protein(such as, for example, human serum albumin or another serum protein suchas IgG), so as to provide increased half-life in serum. Such amino acidsequences for example include the Nanobodies described below, as well asthe small peptides and binding proteins described in WO 91/01743, WO01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41);4926-42, 2005, as well as to EP 0 368 684, as well as to WO 08/028977,WO 08/043821, WO 08/043822 by Ablynx N.V. and US provisional applicationof Ablynx N.V. entitled “Peptides capable of binding to serum proteins”filed on Dec. 5, 2006 ((see also PCT/EP2007/063348).

Such amino acid sequences may in particular be directed against serumalbumin (and more in particular human serum albumin) and/or against IgG(and more in particular human IgG). For example, such amino acidsequences may be amino acid sequences that are directed against (human)serum albumin and amino acid sequences that can bind to amino acidresidues on (human) serum albumin that are not involved in binding ofserum albumin to FcRn (see for example WO 06/0122787) and/or amino acidsequences that are capable of binding to amino acid residues on serumalbumin that do not form part of domain III of serum albumin (see againfor example WO 06/0122787); amino acid sequences that have or canprovide an increased half-life (see for example WO 08/028977 by AblynxN.V.); amino acid sequences against human serum albumin that arecross-reactive with serum albumin from at least one species of mammal,and in particular with at least one species of primate (such as, withoutlimitation, monkeys from the genus Macaca (such as, and in particular,cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta)) and baboon (Papio ursinus), reference is again made to WO08/028977; amino acid sequences that can bind to serum albumin in a pHindependent manner (see for example WO 08/043821 by Ablynx entitled“Amino acid sequences that bind to serum proteins in a manner that isessentially independent of the pH, compounds comprising the same, anduses thereof”) and/or amino acid sequences that are conditional binders(see for example WO 08/043822 by Ablynx N.V. entitled “Amino acidsequences that bind to a desired molecule in a conditional manner”).

According to another aspect, the one or more further amino acidsequences may comprise one or more parts, fragments or domains ofconventional 4-chain antibodies (and in particular human antibodies)and/or of heavy chain antibodies. For example, although usually lesspreferred, a Nanobody of the invention may be linked to a conventional(preferably human) V_(H) or V_(L) domain or to a natural or syntheticanalog of a V_(H) or V_(L) domain, again optionally via a linkersequence (including but not limited to other (single) domain antibodies,such as the dAb's described by Ward et al.).

The at least one Nanobody may also be linked to one or more (preferablyhuman) C_(H)1, C_(H)2 and/or C_(H)3 domains, optionally via a linkersequence. For instance, a Nanobody linked to a suitable C_(H)1 domaincould for example be used—together with suitable light chains—togenerate antibody fragments/structures analogous to conventional Fabfragments or F(ab′)₂ fragments, but in which one or (in case of anF(ab′)₂ fragment) one or both of the conventional V_(H) domains havebeen replaced by a Nanobody of the invention. Also, two Nanobodies couldbe linked to a C_(H)3 domain (optionally via a linker) to provide aconstruct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, oneor more Nanobodies of the invention may be linked (optionally via asuitable linker or hinge region) to one or more constant domains (forexample, 2 or 3 constant domains that can be used as part of/to form anFc portion), to an Fc portion and/or to one or more antibody parts,fragments or domains that confer one or more effector functions to thepolypeptide of the invention and/of may confer the ability to bind toone or more Fc receptors. For example, for this purpose, and withoutbeing limited thereto, the one or more further amino acid sequences maycomprise one or more C_(H)2 and/or C_(H)3 domains of an antibody, suchas from a heavy chain antibody (as described herein) and more preferablyfrom a conventional human 4-chain antibody; and/or may form (part of)and Fc region, for example from IgG (e.g. from IgG1, IgG2, IgG3 orIgG4), from IgE or from another human Ig such as IgA, IgD or IgM. Forexample, WO 94/04678 describes heavy chain antibodies comprising aCamelid V_(HH) domain or a humanized derivative thereof (i.e. aNanobody), in which the Camelidae C₁₋₁₂ and/or C_(H)3 domain have beenreplaced by human C_(H)2 and C_(H)3 domains, so as to provide animmunoglobulin that consists of 2 heavy chains each comprising aNanobody and human C_(H)2 and C_(H)3 domains (but no C_(H)1 domain),which immunoglobulin has the effector function provided by the C_(H)2and C_(H)3 domains and which immunoglobulin can function without thepresence of any light chains. Other amino acid sequences that can besuitably linked to the Nanobodies of the invention so as to provide aneffector function will be clear to the skilled person, and may be chosenon the basis of the desired effector function(s). Reference is forexample made to WO 04/058820, WO 99/42077, WO 02/056910 and WO05/017148, as well as the review by Holliger and Hudson, supra; and tothe non-prepublished US provisional application by Ablynx N.V. entitled“Constructs comprising single variable domains and an Fc portion derivedfrom IgE” which has a filing date of Dec. 4, 2007. Coupling of aNanobody of the invention to an Fc portion may also lead to an increasedhalf-life, compared to the corresponding Nanobody of the invention. Forsome applications, the use of an Fc portion and/or of constant domains(i.e. C_(H)2 and/or C_(H)3 domains) that confer increased half-lifewithout any biologically significant effector function may also besuitable or even preferred. Other suitable constructs comprising one ormore Nanobodies and one or more constant domains with increasedhalf-life in vivo will be clear to the skilled person, and may forexample comprise two Nanobodies linked to a C_(H)3 domain, optionallyvia a linker sequence. Generally, any fusion protein or derivatives withincreased half-life will preferably have a molecular weight of more than50 kD, the cut-off value for renal absorption.

In another one specific, but non-limiting, aspect, in order to form apolypeptide of the invention, one or more amino acid sequences of theinvention may be linked (optionally via a suitable linker or hingeregion) to naturally occurring, synthetic or semisynthetic constantdomains (or analogs, variants, mutants, parts or fragments thereof) thathave a reduced (or essentially no) tendency to self-associate intodimers (i.e. compared to constant domains that naturally occur inconventional 4-chain antibodies). Such monomeric (i.e. notself-associating) Fc chain variants, or fragments thereof, will be clearto the skilled person. For example, Helm et al., J Biol Chem 1996 2717494, describe monomeric Fcs chain variants that can be used in thepolypeptide chains of the invention.

Also, such monomeric Fc chain variants are preferably such that they arestill capable of binding to the complement or the relevant Fcreceptor(s) (depending on the Fc portion from which they are derived),and/or such that they still have some or all of the effector functionsof the Fc portion from which they are derived (or at a reduced levelstill suitable for the intended use). Alternatively, in such apolypeptide chain of the invention, the monomeric Fc chain may be usedto confer increased half-life upon the polypeptide chain, in which casethe monomeric Fc chain may also have no or essentially no effectorfunctions.

Bivalent/multivalent, bispecific/multispecific orbiparatopic/multiparatopic polypeptides of the invention may also belinked to Fc portions, in order to provide polypeptide constructs of thetype that is described in the non-prepublished U.S. provisionalapplication 61/005,331 entitled “immunoglobulin constructs” filed onDec. 4, 2007.

The further amino acid sequences may also form a signal sequence orleader sequence that directs secretion of the Nanobody or thepolypeptide of the invention from a host cell upon synthesis (forexample to provide a pre-, pro- or prepro-form of the polypeptide of theinvention, depending on the host cell used to express the polypeptide ofthe invention).

The further amino acid sequence may also form a sequence or signal thatallows the Nanobody or polypeptide of the invention to be directedtowards and/or to penetrate or enter into specific organs, tissues,cells, or parts or compartments of cells, and/or that allows theNanobody or polypeptide of the invention to penetrate or cross abiological barrier such as a cell membrane, a cell layer such as a layerof epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Suitable examples of such amino acid sequences willbe clear to the skilled person, and for example include, but are notlimited to, those mentioned on page 118 of WO 08/020,079. For someapplications, in particular for those applications in which it isintended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation of such acell, the Nanobodies of the invention may also be linked to a(cyto)toxic protein or polypeptide. Examples of such toxic proteins andpolypeptides which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic polypeptide of the invention will beclear to the skilled person and can for example be found in the priorart cited above and/or in the further description herein. One example isthe so-called ADEPT™ technology described in WO 03/055527.

According to one preferred, but non-limiting aspect, said one or morefurther amino acid sequences comprise at least one further Nanobody, soas to provide a polypeptide of the invention that comprises at leasttwo, such as three, four, five or more Nanobodies, in which saidNanobodies may optionally be linked via one or more linker sequences (asdefined herein). As described on pages 119 and 120 of WO 08/020,079,polypeptides of the invention that comprise two or more Nanobodies, ofwhich at least one is a Nanobody of the invention, will also be referredto herein as “multivalent” polypeptides of the invention, and theNanobodies present in such polypeptides will also be referred to hereinas being in a “multivalent format”. For example, “bivalent” and“trivalent” polypeptides of the invention may be as further described onpages 119 and 120 of WO 08/020,079.

Polypeptides of the invention that contain at least two Nanobodies, inwhich at least one Nanobody is directed against a first antigen (i.e.against multiscavenger receptors,) and at least one Nanobody is directedagainst a second antigen (i.e. different from multiscavengerreceptors,), will also be referred to as “multispecific” polypeptides ofthe invention, and the Nanobodies present in such polypeptides will alsobe referred to herein as being in a “multispecific format”. Thus, forexample, a “bispecific” polypeptide of the invention is a polypeptidethat comprises at least one Nanobody directed against a first antigen(i.e. multiscavenger receptors,) and at least one further Nanobodydirected against a second antigen (i.e. different from multiscavengerreceptors,), whereas a “trispecific” polypeptide of the invention is apolypeptide that comprises at least one Nanobody directed against afirst antigen (i.e. multiscavenger receptors,), at least one furtherNanobody directed against a second antigen (i.e. different frommultiscavenger receptors,) and at least one further Nanobody directedagainst a third antigen (i.e. different from both multiscavengerreceptors, and the second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first Nanobody directed against multiscavengerreceptors, and a second Nanobody directed against a second antigen, inwhich said first and second Nanobody may optionally be linked via alinker sequence (as defined herein); whereas a trispecific polypeptideof the invention in its simplest form is a trivalent polypeptide of theinvention (as defined herein), comprising a first Nanobody directedagainst multiscavenger receptors, a second Nanobody directed against asecond antigen and a third Nanobody directed against a third antigen, inwhich said first, second and third Nanobody may optionally be linked viaone or more, and in particular one and more, in particular two, linkersequences.

However, as will be clear from the description hereinabove, theinvention is not limited thereto, in the sense that a multispecificpolypeptide of the invention may comprise at least one Nanobody againstmultiscavenger receptors, and any number of Nanobodies directed againstone or more antigens different from multiscavenger receptors.

Furthermore, although it is encompassed within the scope of theinvention that the specific order or arrangement of the variousNanobodies in the polypeptides of the invention may have some influenceon the properties of the final polypeptide of the invention (includingbut not limited to the affinity, specificity or avidity formultiscavenger receptors, or against the one or more other antigens),said order or arrangement is usually not critical and may be suitablychosen by the skilled person, optionally after some limited routineexperiments based on the disclosure herein. Thus, when reference is madeto a specific multivalent or multispecific polypeptide of the invention,it should be noted that this encompasses any order or arrangements ofthe relevant Nanobodies, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that thepolypeptides of the invention contain two or more Nanobodies and one ormore further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or moreV_(HH) domains and their preparation, reference is also made to Conrathet al., J. Biol. Chem. Vol. 276, 10. 7346-7350, 2001; Muyldermans,Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to forexample WO 96/34103 and WO 99/23221. Some other examples of somespecific multispecific and/or multivalent polypeptide of the inventioncan be found in the applications by Ablynx N.V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptideof the invention comprises at least one Nanobody of the invention and atleast one Nanobody that provides for an increased half-life. SuchNanobodies may for example be Nanobodies that are directed against aserum protein, and in particular a human serum protein, such as humanserum albumin, thyroxine-binding protein, (human) transferrin,fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one ofthe serum proteins listed in WO 04/003019. Of these, Nanobodies that canbind to serum albumin (and in particular human serum albumin) or to IgG(and in particular human IgG, see for example Nanobody VH-1 described inthe review by Muyldermans, supra) are particularly preferred (althoughfor example, for experiments in mice or primates, Nanobodies against orcross-reactive with mouse serum albumin (MSA) or serum albumin from saidprimate, respectively, can be used. However, for pharmaceutical use,Nanobodies against human serum albumin or human IgG will usually bepreferred). Nanobodies that provide for increased half-life and that canbe used in the polypeptides of the invention include the Nanobodiesdirected against serum albumin that are described in WO 04/041865, in WO06/122787 and in the further patent applications by Ablynx N.V., such asthose mentioned above.

For example, the some preferred Nanobodies that provide for increasedhalf-life for use in the present invention include Nanobodies that canbind to amino acid residues on (human) serum albumin that are notinvolved in binding of serum albumin to FeRn (see for example WO06/0122787); Nanobodies that are capable of binding to amino acidresidues on serum albumin that do not form part of domain III of serumalbumin (see for example WO 06/0122787); Nanobodies that have or canprovide an increased half-life (see for example WO 08/028977 by AblynxN.V mentioned herein); Nanobodies against human serum albumin that arecross-reactive with serum albumin from at least one species of mammal,and in particular with at least one species of primate (such as, withoutlimitation, monkeys from the genus Macaca (such as, and in particular,cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta)) and baboon (Papio ursinus)) (see for example WO 08/028977 byAblynx N.V)); Nanobodies that can bind to serum albumin in a pHindependent manner (see for example WO2008/043821 by Ablynx N.V.mentioned herein) and/or Nanobodies that are conditional binders (seefor example WO 08/043822 by Ablynx N.V.).

Some particularly preferred Nanobodies that provide for increasedhalf-life and that can be used in the polypeptides of the inventioninclude the Nanobodies ALB-1 to ALB-10 disclosed in WO 06/122787 (seeTables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787) isparticularly preferred.

According to a specific, but non-limiting aspect of the invention, thepolypeptides of the invention contain, besides the one or moreNanobodies of the invention, at least one Nanobody against human serumalbumin.

Generally, any polypeptides of the invention with increased half-lifethat contain one or more Nanobodies of the invention, and anyderivatives of Nanobodies of the invention or of such polypeptides thathave an increased half-life, preferably have a half-life that is atleast 1.5 times, preferably at least 2 times, such as at least 5 times,for example at least 10 times or more than 20 times, greater than thehalf-life of the corresponding Nanobody of the invention per se. Forexample, such a derivative or polypeptides with increased half-life mayhave a half-life that is increased with more than 1 hours, preferablymore than 2 hours, more preferably more than 6 hours, such as more than12 hours, or even more than 24, 48 or 72 hours, compared to thecorresponding Nanobody of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchderivatives or polypeptides may exhibit a serum half-life in human of atleast about 12 hours, preferably at least 24 hours, more preferably atleast 48 hours, even more preferably at least 72 hours or more. Forexample, such derivatives or polypeptides may have a half-life of atleast 5 days (such as about 5 to 10 days), preferably at least 9 days(such as about 9 to 14 days), more preferably at least about 10 days(such as about 10 to 15 days), or at least about 11 days (such as about11 to 16 days), more preferably at least about 12 days (such as about 12to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to one aspect of the invention the polypeptides are capable ofbinding to one or more molecules which can increase the half-life of thepolypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and theirhalf-life increased by binding to molecules which resist degradationand/or clearance or sequestration. Typically, such molecules arenaturally occurring proteins which themselves have a long half-life invivo.

Another preferred, but non-limiting example of a multispecificpolypeptide of the invention comprises at least one Nanobody of theinvention and at least one Nanobody that directs the polypeptide of theinvention towards, and/or that allows the polypeptide of the inventionto penetrate or to enter into specific organs, tissues, cells, or partsor compartments of cells, and/or that allows the Nanobody to penetrateor cross a biological barrier such as a cell membrane, a cell layer suchas a layer of epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Examples of such Nanobodies include Nanobodies thatare directed towards specific cell-surface proteins, markers or epitopesof the desired organ, tissue or cell (for example cell-surface markersassociated with tumor cells), and the single-domain brain targetingantibody fragments described in WO 02/057445 and WO 06/040153, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

In the polypeptides of the invention, the one or more Nanobodies and theone or more polypeptides may be directly linked to each other (as forexample described in WO 99/23221) and/or may be linked to each other viaone or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecificpolypeptides will be clear to the skilled person, and may generally beany linker or spacer used in the art to link amino acid sequences.Preferably, said linker or spacer is suitable for use in constructingproteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itsshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(u) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each Nanobody by itselfforms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 50, preferably between1 and 30, such as between 1 and 10 amino acid residues. Some preferredexamples of such amino acid sequences include gly-ser linkers, forexample of the type (gly_(x)ser_(y))_(z), such as (for example(gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and the GS30,GS15, GS9 and GS7 linkers described in the applications by Ablynxmentioned herein (see for example WO 06/040153 and WO 06/122825), aswell as hinge-like regions, such as the hinge regions of naturallyoccurring heavy chain antibodies or similar sequences (such as describedin WO 94/04678).

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) andGS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, polyethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for multiscavenger receptors, or for one or moreof the other antigens. Based on the disclosure herein, the skilledperson will be able to determine the optimal linker(s) for use in aspecific polypeptide of the invention, optionally after some limitedroutine experiments.

For example, in multivalent polypeptides of the invention that compriseNanobodies directed against a multimeric antigen (such as a multimericreceptor or other protein), the length and flexibility of the linker arepreferably such that it allows each Nanobody of the invention present inthe polypeptide to bind to the antigenic determinant on each of thesubunits of the multimer. Similarly, in a multispecific polypeptide ofthe invention that comprises Nanobodies directed against two or moredifferent antigenic determinants on the same antigen (for exampleagainst different epitopes of an antigen and/or against differentsubunits of a multimeric receptor, channel or protein), the length andflexibility of the linker are preferably such that it allows eachNanobody to bind to its intended antigenic determinant. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linker(s) for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the Nanobodies of theinvention). For example, linkers containing one or more charged aminoacid residues (see Table A-2 on page 48 of the International applicationWO 08/020,079) can provide improved hydrophilic properties, whereaslinkers that form or contain small epitopes or tags can be used for thepurposes of detection, identification and/or purification. Again, basedon the disclosure herein, the skilled person will be able to determinethe optimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thereto. For example, when a polypeptideof the invention comprises three of more Nanobodies, it is possible tolink them by use of a linker with three or more “arms”, which each “arm”being linked to a Nanobody, so as to provide a “star-shaped” construct.It is also possible, although usually less preferred, to use circularconstructs.

The invention also comprises derivatives of the polypeptides of theinvention, which may be essentially analogous to the derivatives of theNanobodies of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that “essentiallyconsist” of a polypeptide of the invention (in which the wording“essentially consist of” has essentially the same meaning as indicatedhereinabove).

According to one aspect of the invention, the polypeptide of theinvention is in essentially isolated from, as defined herein.

The amino acid sequences, Nanobodies, polypeptides and nucleic acids ofthe invention can be prepared in a manner known per se, as will be clearto the skilled person from the further description herein. For example,the Nanobodies and polypetides of the invention can be prepared in anymanner known per se for the preparation of antibodies and in particularfor the preparation of antibody fragments (including but not limited to(single) domain antibodies and ScFv fragments). Some preferred, butnon-limiting methods for preparing the amino acid sequences, Nanobodies,polypeptides and nucleic acids include the methods and techniquesdescribed herein.

As will be clear to the skilled person, one particularly useful methodfor preparing an amino acid sequence, Nanobody and/or a polypeptide ofthe invention generally comprises the steps of:

-   i) the expression, in a suitable host cell or host organism (also    referred to herein as a “host of the invention”) or in another    suitable expression system of a nucleic acid that encodes said amino    acid sequence, Nanobody or polypeptide of the invention (also    referred to herein as a “nucleic acid of the invention”), optionally    followed by:-   ii) isolating and/or purifying the amino acid sequence, Nanobody or    polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   i) cultivating and/or maintaining a host of the invention under    conditions that are such that said host of the invention expresses    and/or produces at least one amino acid sequence, Nanobody and/or    polypeptide of the invention; optionally followed by:-   ii) isolating and/or purifying the amino acid sequence, Nanobody or    polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one aspect of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the amino acidsequences for the polypeptides of the invention given herein, and/or canbe isolated from a suitable natural source. To provide analogs,nucleotide sequences encoding naturally occurring V_(HH) domains can forexample be subjected to site-directed mutagenesis, so at to provide anucleic acid of the invention encoding said analog. Also, as will beclear to the skilled person, to prepare a nucleic acid of the invention,also several nucleotide sequences, such as at least one nucleotidesequence encoding a Nanobody and for example nucleic acids encoding oneor more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using for example a sequence of anaturally occurring form of multiscavenger receptors as a template.These and other techniques will be clear to the skilled person, andreference is again made to the standard handbooks, such as Sambrook etal. and Ausubel et al., mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art and as described on pages 131-134 of WO 08/020,079(incorporated herein by reference). Such genetic constructs generallycomprise at least one nucleic acid of the invention that is optionallylinked to one or more elements of genetic constructs known per se, suchas for example one or more suitable regulatory elements (such as asuitable promoter(s), enhancer(s), terminator(s), etc.) and the furtherelements of genetic constructs referred to herein. Such geneticconstructs comprising at least one nucleic acid of the invention willalso be referred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting aspect, a genetic construct of theinvention comprises

-   i) at least one nucleic acid of the invention; operably connected to-   ii) one or more regulatory elements, such as a promoter and    optionally a suitable terminator;    and optionally also-   iii) one or more further elements of genetic constructs known per    se;

in which the terms “operably connected” and “operably linked” have themeaning given on pages 131-134 of WO 08/020,079; and in which the“regulatory elements”, “promoter”, “terminator” and “further elements”are as described on pages 131-134 of WO 08/020,079; and in which thegenetic constructs may further be as described on pages 131-134 of WO08/020,079.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the amino acid sequence, Nanobody orpolypeptide of the invention. Suitable hosts or host cells will be clearto the skilled person, and may for example be any suitable fungal,prokaryotic or eukaryotic cell or cell line or any suitable fungal,prokaryotic or eukaryotic organism, for example those described on pages134 and 135 of WO 08/020,079.; as well as all other hosts or host cellsknown per se for the expression and production of antibodies andantibody fragments (including but not limited to (single) domainantibodies and ScFv fragments), which will be clear to the skilledperson. Reference is also made to the general background art citedhereinabove, as well as to for example WO 94/29457; WO 96/34103; WO99/42077; Frenken et al., (1998), supra; Riechniann and Muyldermans,(1999), supra; van der Linden, (2000), supra; Thomassen et al., (2002),supra; Joosten et al., (2003), supra; Joosten et al., (2005), supra; andthe further references cited herein.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be introduced and expressed in one or more cells, tissues ororgans of a multicellular organism, for example for prophylactic and/ortherapeutic purposes (e.g. as a gene therapy), as further described onpages 135 and 136 of in WO 08/020079 and in the further references citedin WO 08/020079.

For expression of the Nanobodies in a cell, they may also be expressedas so-called “intrabodies”, as for example described in WO 94/02610, WO95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. &Biocca, S. (1997) Intracellular Antibodies: Development andApplications. Landes and Springer-Verlag; and in Kontermann, Methods 34,(2004), 163-170.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan for example also be produced in the milk of transgenic mammals, forexample in the milk of rabbits, cows, goats or sheep (see for exampleU.S. Pat. Nos. 6,741,957, 6,304,489 and 6,849,992 for general techniquesfor introducing transgenes into mammals), in plants or parts of plantsincluding but not limited to their leaves, flowers, fruits, seed, rootsor turbers (for example in tobacco, maize, soybean or alfalfa) or in forexample pupae of the silkworm Bombix mori.

Furthermore, the amino acid sequences, Nanobodies and polypeptides ofthe invention can also be expressed and/or produced in cell-freeexpression systems, and suitable examples of such systems will be clearto the skilled person. Some preferred, but non-limiting examples includeexpression in the wheat germ system; in rabbit reticulocyte lysates; orin the E. coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies isthat the polypeptides based thereon can be prepared through expressionin a suitable bacterial system, and suitable bacterial expressionsystems, vectors, host cells, regulatory elements, etc., will be clearto the skilled person, for example from the references cited above. Itshould however be noted that the invention in its broadest sense is notlimited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of Nanobodies or Nanobody-containing proteintherapeutics include strains of E. coli, Pichia pastoris, S. cerevisiaethat are suitable for large scale expression/production/fermentation,and in particular for large scale pharmaceutical (i.e. GMP grade)expression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a Nanobody-containingrecombinant protein for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression. Preferably, either a humancell or cell line is used (i.e. leading to a protein that essentiallyhas a human glycosylation pattern) or another mammalian cell line isused that can provide a glycosylation pattern that is essentially and/orfunctionally the same as human glycosylation or at least mimics humanglycosylation. Generally, prokaryotic hosts such as E. coli do not havethe ability to glycosylate proteins, and the use of lower eukaryotessuch as yeast usually leads to a glycosylation pattern that differs fromhuman glycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired amino acid sequence, Nanobody orpolypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention is glycosylated.According to another non-limiting aspect of the invention, the aminoacid sequence, Nanobody or polypeptide of the invention isnon-glycosylated.

According to one preferred, but non-limiting aspect of the invention,the amino acid sequence, Nanobody or polypeptide of the invention isproduced in a bacterial cell, in particular a bacterial cell suitablefor large scale pharmaceutical production, such as cells of the strainsmentioned above.

According to another preferred, but non-limiting aspect of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a yeast cell, in particular a yeast cellsuitable for large scale pharmaceutical production, such as cells of thespecies mentioned above.

According to yet another preferred, but non-limiting aspect of theinvention, the amino acid sequence, Nanobody or polypeptide of theinvention is produced in a mammalian cell, in particular in a human cellor in a cell of a human cell line, and more in particular in a humancell or in a cell of a human cell line that is suitable for large scalepharmaceutical production, such as the cell lines mentioned hereinabove.

As further described on pages 138 and 139 of WO 08/020,079, whenexpression in a host cell is used to produce the amino acid sequences,Nanobodies and the polypeptides of the invention, the amino acidsequences, Nanobodies and polypeptides of the invention can be producedeither intracellullarly (e.g. in the cytosol, in the periplasma or ininclusion bodies) and then isolated from the host cells and optionallyfurther purified; or can be produced extracellularly (e.g. in the mediumin which the host cells are cultured) and then isolated from the culturemedium and optionally further purified. Thus, according to onenon-limiting aspect of the invention, the amino acid sequence, Nanobodyor polypeptide of the invention is an amino acid sequence, Nanobody orpolypeptide that has been produced intracellularly and that has beenisolated from the host cell, and in particular from a bacterial cell orfrom an inclusion body in a bacterial cell. According to anothernon-limiting aspect of the invention, the amino acid sequence, Nanobodyor polypeptide of the invention is an amino acid sequence, Nanobody orpolypeptide that has been produced extracellularly, and that has beenisolated from the medium in which the host cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude those mentioned on pages 139 and 140 of WO 08/020,079.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include those mentioned on page 140 of WO 08/020,079.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an amino acid sequence, Nanobody or polypeptide of theinvention (and in case of a host organism: in at least one cell, part,tissue or organ thereof). The invention also includes furthergenerations, progeny and/or offspring of the host cell or host organismof the invention, that may for instance be obtained by cell division orby sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of theinvention, the transformed host cell or transformed host organism maygenerally be kept, maintained and/or cultured under conditions such thatthe (desired) amino acid sequence, Nanobody or polypeptide of theinvention is expressed/produced. Suitable conditions will be clear tothe skilled person and will usually depend upon the host cell/hostorganism used, as well as on the regulatory elements that control theexpression of the (relevant) nucleotide sequence of the invention.Again, reference is made to the handbooks and patent applicationsmentioned above in the paragraphs on the genetic constructs of theinvention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acidsequence, Nanobody or polypeptide of the invention may (first) begenerated in an immature form (as mentioned above), which may then besubjected to post-translational modification, depending on the hostcell/host organism used. Also, the amino acid sequence, Nanobody orpolypeptide of the invention may be glycosylated, again depending on thehost cell/host organism used.

The amino acid sequence, Nanobody or polypeptide of the invention maythen be isolated from the host cell/host organism and/or from the mediumin which said host cell or host organism was cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the amino acidsequence, Nanobody or polypeptide of the invention) and/or preparativeimmunological techniques (i.e. using antibodies against the amino acidsequence to be isolated).

Generally, for phatinaceutical use, the polypeptides of the inventionmay be formulated as a pharmaceutical preparation or compositionscomprising at least one polypeptide of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. By means of non-limiting examples, such aformulation may be in a form suitable for oral administration, forparenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described herein.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one Nanobody of the invention or at least one polypeptide of theinvention and at least one suitable carrier, diluent or excipient (i.e.suitable for pharmaceutical use), and optionally one or more furtheractive substances.

Generally, the amino acid sequences, Nanobodies and polypeptides of theinvention can be formulated and administered in any suitable mannerknown per se, for which reference is for example made to the generalbackground art cited above (and in particular to WO 04/041862, WO04/041863, WO 04/041865, WO 04/041867 and WO 08/020,079) as well as tothe standard handbooks, such as Remington's Pharmaceutical Sciences,18^(th) Ed., Mack Publishing Company, USA (1990), Remington, the Scienceand Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins(2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.),Wiley, Weinheim, 2007 (see for example pages 252-255).

For example, the amino acid sequences, Nanobodies and polypeptides ofthe invention may be formulated and administered in any manner known perse for conventional antibodies and antibody fragments (including ScFv'sand diabodies) and other pharmaceutically active proteins. Suchformulations and methods for preparing the same will be clear to theskilled person, and for example include preparations suitable forparenteral administration (for example intravenous, intraperitoneal,subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecaladministration) or for topical (i.e. transdermal or intradermal)administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, those mentioned onpage 143 of WO 08/020,079. Usually, aqueous solutions or suspensionswill be preferred.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be administered using gene therapy methods of delivery. See,e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in itsentirety. Using a gene therapy method of delivery, primary cellstransfected with the gene encoding an amino acid sequence, Nanobody orpolypeptide of the invention can additionally be transfected with tissuespecific promoters to target specific organs, tissue, grafts, tumors, orcells and can additionally be transfected with signal and stabilizationsequences for subcellularly localized expression.

Thus, the amino acid sequences, Nanobodies and polypeptides of theinvention may be systemically administered, e.g., orally, in combinationwith a pharmaceutically acceptable vehicle such as an inert diluent oran assimilable edible carrier. They may be enclosed in hard or softshell gelatin capsules, may be compressed into tablets, or may beincorporated directly with the food of the patient's diet. For oraltherapeutic administration, the amino acid sequences, Nanobodies andpolypeptides of the invention may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of theamino acid sequence, Nanobody or polypeptide of the invention. Theirpercentage in the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the amino acidsequence, Nanobody or polypeptide of the invention in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

The tablets, troches, pills, capsules, and the like may also containbinders, excipients, disintegrating agents, lubricants and sweetening orflavouring agents, for example those mentioned on pages 143-144 of WO08/020,079. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the amino acid sequences, Nanobodies and polypeptidesof the invention, sucrose or fructose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor. Of course, any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the amino acid sequences,Nanobodies and polypeptides of the invention may be incorporated intosustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The amino acid sequences, Nanobodies and polypeptides of the inventionmay also be administered intravenously or intraperitoneally by infusionor injection, as further described on pages 144 and 145 of WO08/020,079.

For topical administration, the amino acid sequences, Nanobodies andpolypeptides of the invention may be applied in pure form, i.e., whenthey are liquids. However, it will generally be desirable to administerthem to the skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid,as further described on page 145 of WO 08/020,079.

Generally, the concentration of the amino acid sequences, Nanobodies andpolypeptides of the invention in a liquid composition, such as a lotion,will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. Theconcentration in a semi-solid or solid composition such as a gel or apowder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the amino acid sequences, Nanobodies and polypeptides ofthe invention required for use in treatment will vary not only with theparticular amino acid sequence, Nanobody or polypeptide selected butalso with the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician. Also thedosage of the amino acid sequences, Nanobodies and polypeptides of theinvention varies depending on the target cell, tumor, tissue, graft, ororgan.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one a disease wherein multiscanvengerreceptors are implicated, said method comprising administering, to asubject in need thereof, a pharmaceutically active amount of an aminoacid sequence of the invention, of a Nanobody of the invention, of apolypeptide of the invention, and/or of a pharmaceutical compositioncomprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with multiscavengerreceptors, with its biological or pharmacological activity, and/or withthe biological pathways or signalling in which multiscavenger receptorsis involved, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of an amino acid sequence ofthe invention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can be treatedby modulating multiscavenger receptors, its biological orpharmacological activity, and/or the biological pathways or signallingin which multiscavenger receptors is involved, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of an amino acid sequence of the invention, of a Nanobody of theinvention, of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same. In particular, said pharmaceuticallyeffective amount may be an amount that is sufficient to modulatemultiscavenger receptors, its biological or pharmacological activity,and/or the biological pathways or signalling in which multiscavengerreceptors is involved; and/or an amount that provides a level of theamino acid sequence of the invention, of a Nanobody of the invention, ofa polypeptide of the invention in the circulation that is sufficient tomodulate multiscavenger receptors, its biological or pharmacologicalactivity, and/or the biological pathways or signalling in whichmultiscavenger receptors is involved.

The invention furthermore relates to a method for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering an amino acid sequence of the invention,a Nanobody of the invention or a polypeptide of the invention to apatient, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of an amino acid sequence ofthe invention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy,and in particular for passive immunotherapy, which method comprisesadministering, to a subject suffering from or at risk of the diseasesand disorders mentioned herein, a pharmaceutically active amount of anamino acid sequence of the invention, of a Nanobody of the invention, ofa polypeptide of the invention, and/or of a pharmaceutical compositioncomprising the same.

In the above methods, the amino acid sequences, Nanobodies and/orpolypeptides of the invention and/or the compositions comprising thesame can be administered in any suitable manner, depending on thespecific pharmaceutical formulation or composition to be used. Thus, theamino acid sequences, Nanobodies and/or polypeptides of the inventionand/or the compositions comprising the same can for example beadministered orally, intraperitoneally (e.g. intravenously,subcutaneously, intramuscularly, or via any other route ofadministration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorswell known to the clinician.

The amino acid sequences, Nanobodies and/or polypeptides of theinvention and/or the compositions comprising the same are administeredaccording to a regime of treatment that is suitable for preventingand/or treating the disease or disorder to be prevented or treated. Theclinician will generally be able to determine a suitable treatmentregimen, depending on factors such as the disease or disorder to beprevented or treated, the severity of the disease to be treated and/orthe severity of the symptoms thereof, the specific amino acid sequence,Nanobody or polypeptide of the invention to be used, the specific routeof administration and pharmaceutical formulation or composition to beused, the age, gender, weight, diet, general condition of the patient,and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, Nanobodies and/or polypeptides of theinvention, or of one or more compositions comprising the same, in one ormore pharmaceutically effective amounts or doses. The specific amount(s)or doses to administered can be determined by the clinician, again basedon the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific amino acid sequence,Nanobody and polypeptide of the invention to be used, the specific routeof administration and the specific pharmaceutical formulation orcomposition used, the amino acid sequences. Nanobodies and polypeptidesof the invention will generally be administered in an amount between 1gram and 0.01 microgram per kg body weight per day, preferably between0.1 gram and 0.1 microgram per kg body weight per day, such as about 1,10, 100 or 1000 microgram per kg body weight per day, eithercontinuously (e.g. by infusion), as a single daily dose or as multipledivided doses during the day. The clinician will generally be able todetermine a suitable daily dose, depending on the factors mentionedherein. It will also be clear that in specific cases, the clinician maychoose to deviate from these amounts, for example on the basis of thefactors cited above and his expert judgment. Generally, some guidance onthe amounts to be administered can be obtained from the amounts usuallyadministered for comparable conventional antibodies or antibodyfragments against the same target administered via essentially the sameroute, taking into account however differences in affinity/avidity,efficacy, biodistribution, half-life and similar factors well known tothe skilled person.

Usually, in the above method, a single amino acid sequence, Nanobody orpolypeptide of the invention will be used. It is however within thescope of the invention to use two or more amino acid sequences,Nanobodies and/or polypeptides of the invention in combination.

The Nanobodies, amino acid sequences and polypeptides of the inventionmay also be used in combination with one or more furtherpharmaceutically active compounds or principles, i.e. as a combinedtreatment regimen, which may or may not lead to a synergistic effect.Again, the clinician will be able to select such further compounds orprinciples, as well as a suitable combined treatment regimen, based onthe factors cited above and his expert judgement.

In particular, the amino acid sequences, Nanobodies and polypeptides ofthe invention may be used in combination with other pharmaceuticallyactive compounds or principles that are or can be used for theprevention and/or treatment of the diseases and disorders cited herein,as a result of which a synergistic effect may or may not be obtained.Examples of such compounds and principles, as well as routes, methodsand pharmaceutical formulations or compositions for administering themwill be clear to the clinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for prevention and/or treatment of at leastone a disease wherein multiscanvenger receptors are implicated; and/orfor use in one or more of the methods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention in the preparation of apharmaceutical composition for the prevention and/or treatment of atleast one disease or disorder that can be prevented and/or treated byadministering an amino acid sequence, Nanobody or polypeptide of theinvention to a patient.

More in particular, the invention relates to the use of an amino acidsequence, Nanobody or polypeptide of the invention in the preparation ofa pharmaceutical composition for the prevention and/or treatment of adisease wherein multiscanvenger receptors are implicated, and inparticular for the prevention and treatment of one or more of thediseases and disorders listed herein.

Again, in such a pharmaceutical composition, the one or more amino acidsequences, Nanobodies or polypeptides of the invention may also besuitably combined with one or more other active principles, such asthose mentioned herein.

Finally, although the use of the Nanobodies of the invention (as definedherein) and of the polypeptides of the invention is much preferred, itwill be clear that on the basis of the description herein, the skilledperson will also be able to design and/or generate, in an analogousmanner, other amino acid sequences and in particular (single) domainantibodies against multiscavenger receptors, as well as polypeptidescomprising such (single) domain antibodies.

For example, it will also be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's mentioned above for theNanobodies of the invention onto such (single) domain antibodies orother protein scaffolds, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting will be clear to the skilled person and are well known inthe art, see for example those mentioned in WO 08/020,079. For example,techniques known per se for grafting mouse or rat CDR's onto humanframeworks and scaffolds can be used in an analogous manner to providechimeric proteins comprising one or more of the CDR's of the Nanobodiesof the invention and one or more human framework regions or sequences.

It should also be noted that, when the Nanobodies of the inventionscontain one or more other CDR sequences than the preferred. CDRsequences mentioned above, these CDR sequences can be obtained in anymanner known per se, for example using one or more of the techniquesdescribed in WO 08/020,079.

Further uses of the amino acid sequences, Nanobodies, polypeptides,nucleic acids, genetic constructs and hosts and host cells of theinvention will be clear to the skilled person based on the disclosureherein. For example, and without limitation, the amino acid sequences ofthe invention can be linked to a suitable carrier or solid support so asto provide a medium than can be used in a manner known per se to purifymultiscavenger receptors from compositions and preparations comprisingthe same. Derivatives of the amino acid sequences of the invention thatcomprise a suitable detectable label can also be used as markers todetermine (qualitatively or quantitatively) the presence ofmultiscavenger receptors in a composition or preparation or as a markerto selectively detect the presence of multiscavenger receptors on thesurface of a cell or tissue (for example, in combination with suitablecell sorting techniques).

Preferred Aspects:

-   Aspect A-1: An amino acid sequence that is directed against and/or    that can specifically bind to at least one of the multiscavenger    receptors.-   Aspect A-2: An amino acid sequence according to aspect A-1, that is    in essentially isolated form.-   Aspect A-3: An amino acid sequence according to aspect A-1 or A-2,    for administration to a subject, wherein said amino acid sequence    does not naturally occur in said subject.-   Aspect A-4: An amino acid sequence that can specifically bind to    multiscavenger receptors with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.    Such an amino acid sequence may in particular be an amino acid    sequence according to any of the preceding aspects.-   Aspect A-5: An amino acid sequence that can specifically bind to    multiscavenger receptors with a rate of association (k_(on)-rate) of    between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷    M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹. Such an amino    acid sequence may in particular be an amino acid sequence according    to any of the preceding aspects.-   Aspect A-6: An amino acid sequence that can specifically bind to    multiscavenger receptors with a rate of dissociation (k_(off) rate)    between 1 s⁻¹ and 10⁻⁶ s⁻¹, preferably between 10⁻² s⁻¹ and 10⁶ s⁻¹,    more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴    s⁻¹ and 10⁻⁶ s⁻¹. Such an amino acid sequence may in particular be    an amino acid sequence according to any of the preceding aspects.-   Aspect A-7: An amino acid sequence that can specifically bind to    multiscavenger receptors with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM. Such an amino acid sequence may in particular    be an amino acid sequence according to any of the preceding aspects.-   Aspect A-8: An amino acid sequence according to any of the preceding    aspects, that is a naturally occurring amino acid sequence (from any    suitable species) or a synthetic or semi-synthetic amino acid    sequence.-   Aspect A-9: An amino acid sequence according to any of the preceding    aspects, that comprises an immunoglobulin fold or that under    suitable conditions is capable of forming an immunoglobulin fold.-   Aspect A-10: An amino acid sequence according to any of the    preceding aspects, that essentially consists of 4 framework regions    (FR1 to FR4 respectively) and 3 complementarity determining regions    (CDR1 to CDR3 respectively).-   Aspect A-11: An amino acid sequence according to any of the    preceding aspects, that is an immunoglobulin sequence.-   Aspect A-12: An amino acid sequence according to any of the    preceding aspects, that is a naturally occurring immunoglobulin    sequence (from any suitable species) or a synthetic or    semi-synthetic immunoglobulin sequence.-   Aspect A-13: An amino acid sequence according to any of the    preceding aspects that is a humanized immunoglobulin sequence, a    camelized immunoglobulin sequence or an immunoglobulin sequence that    has been obtained by techniques such as affinity maturation.-   Aspect A-14: An amino acid sequence according to any of the    preceding aspects, that essentially consists of a light chain    variable domain sequence (e.g. a VL-sequence); or of a heavy chain    variable domain sequence (e.g. a VH-sequence).-   Aspect A-15: An amino acid sequence according to any of the    preceding aspects, that essentially consists of a heavy chain    variable domain sequence that is derived from a conventional    four-chain antibody or that essentially consist of a heavy chain    variable domain sequence that is derived from heavy chain antibody.-   Aspect A-16: An amino acid sequence according to any of the    preceding aspects, that essentially consists of a domain antibody    (or an An amino acid sequence that is suitable for use as a domain    antibody), of a single domain antibody (or an An amino acid sequence    that is suitable for use as a single domain antibody), of a “dAb”    (or an An amino acid sequence that is suitable for use as a dAb) or    of a Nanobody (including but not limited to a VHH sequence).-   Aspect A-17: An amino acid sequence according to any of the    preceding aspects, that essentially consists of a Nanobody.-   Aspect A-18: An amino acid sequence according to any of the    preceding aspects, that essentially consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        An amino acid sequences of SEQ ID NO's: 1 to 22, in which for        the purposes of determining the degree of amino acid identity,        the amino acid residues that form the CDR sequences are        disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect A-19: An amino acid sequence according to any of the    preceding aspects, that essentially consists of a polypeptide that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 308-333, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect A-20: An amino acid sequence according to any of the    preceding aspects, that essentially consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 308-333, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect A-21: An amino acid sequence according to any of the    preceding aspects, that essentially consists of a humanized    Nanobody.-   Aspect A-22: An amino acid sequence according to any of the    preceding aspects, that in addition to the at least one binding site    for binding against multiscavenger receptors, contains one or more    further binding sites for binding against other antigens, proteins    or targets.-   Aspect B-1: An amino acid sequence that is directed against and/or    that can specifically bind multiscavenger receptors, and that    comprises one or more stretches of amino acid residues chosen from    the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 152-177;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 152-177;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 152-177;    -   d) the amino acid sequences of SEQ ID NO's: 204-229;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 204-229;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 204-229;    -   g) the amino acid sequences of SEQ ID NO's: 256-281;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 256-281;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 256-281;    -   or any suitable combination thereof.        Such an amino acid sequence may in particular be an amino acid        sequence according to any of the aspects A-1 to A-22.-   Aspect B-2: An amino acid sequence according to aspect B-1, in which    at least one of said stretches of amino acid residues forms part of    the antigen binding site for binding against multiscavenger    receptors.-   Aspect B-3: An amino acid sequence that is directed against and/or    that can specifically bind multiscavenger receptors and that    comprises two or more stretches of amino acid residues chosen from    the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 152-177;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 152-177;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 152-177;    -   d) the amino acid sequences of SEQ ID NO's: 204-229;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 204-229;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 204-229;    -   g) the amino acid sequences of SEQ ID NO's: 256-281;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 256-281;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 256-281;    -   such that (i) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to        a), b) or c), the second stretch of amino acid residues        corresponds to one of the amino acid sequences according to d),        e), g), h) or i); (ii) when the first stretch of amino acid        residues corresponds to one of the amino acid sequences        according to d), e) or f), the second stretch of amino acid        residues corresponds to one of the amino acid sequences        according to a), b), c), g), h) or i); or (iii) when the first        stretch of amino acid residues corresponds to one of the amino        acid sequences according to g), h) or i), the second stretch of        amino acid residues corresponds to one of the amino acid        sequences according to a), b), c), d), e) or f).        Such an amino acid sequence may in particular be an amino acid        sequence according to any of the aspects A-1 to A-22, B-1 or        B-2.-   Aspect B-4: An amino acid sequence according to aspect B-3, in which    the at least two stretches of amino acid residues forms part of the    antigen binding site for binding against multiscavenger receptors.-   Aspect B-5: An amino acid sequence that is directed against and/or    that can specifically bind multiscavenger receptors and that    comprises three or more stretches of amino acid residues, in which    the first stretch of amino acid residues is chosen from the group    consisting of    -   a) the amino acid sequences of SEQ ID NO's: 152-177;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 152-177;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 152-177;    -   the second stretch of amino acid residues is chosen from the        group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 204-229;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 204-229;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 204-229;    -   and the third stretch of amino acid residues is chosen from the        group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 256-281;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 256-281;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 256-281.        Such an amino acid sequence may in particular be an amino acid        sequence according to any of the aspects A-1 to A-22 and/or B-1        to B-4.-   Aspect B-6: An amino acid sequence according to aspect B-5, in which    the at least three stretches of amino acid residues forms part of    the antigen binding site for binding against multiscavenger    receptors.-   Aspect B-7: An amino acid sequence that is directed against and/or    that can specifically bind multiscavenger receptors in which the CDR    sequences of said amino acid sequence have at least 70% amino acid    identity, preferably at least 80% amino acid identity, more    preferably at least 90% amino acid identity, such as 95% amino acid    identity or more or even essentially 100% amino acid identity with    the CDR sequences of at least one of the amino acid sequences of SEQ    ID NO's: 308-333. Such an amino acid sequence may in particular be    an amino acid sequence according to any of the aspects A-1 to A-22    and/or B-1 to B-6.-   Aspect C-1: An amino acid sequence that is directed against    multiscavenger receptors and that cross-blocks the binding of at    least one of the amino acid sequences of SEQ ID NO's: 308-333 to    multiscavenger receptors. Such an amino acid sequence may in    particular be an amino acid sequence according to any of the aspects    A-1 to A-22 and/or according to aspects B-1 to B-7. Also,    preferably, such an amino acid sequence is able to specifically bind    to multiscavenger receptors.-   Aspect C-2: An amino acid sequence that is directed against    multiscavenger receptors and that is cross-blocked from binding to    multiscavenger receptors by at least one of the amino acid sequences    of SEQ ID NO's: 308-333. Such an amino acid sequence may in    particular be an amino acid sequence according to any of the aspects    A-1 to A-22 and/or according to aspects B-1 to B-7. Also,    preferably, such an amino acid sequence is able to specifically bind    to multiscavenger receptors.-   Aspect C-3: An amino acid sequence according to any of aspects C-1    or C-2, wherein the ability of said amino acid sequence to    cross-block or to be cross-blocked is detected in a Biacore assay.-   Aspect C-4: An amino acid sequence according to any of aspects C-1    to C-3 wherein the ability of said amino acid sequence to    cross-block or to be cross-blocked is detected in an ELISA assay.-   Aspect D-1: An amino acid sequence according to any of aspects B-1    to B-7 or C-1 to C-7, that is in essentially isolated form.-   Aspect D-2: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, and/or D1 for administration to a subject,    wherein said amino acid sequence does not naturally occur in said    subject.-   Aspect D-3: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, and/or D1 to D-2 that can specifically bind to    multiscavenger receptors with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/litre or less, and preferably 10⁻⁷ to 10⁻¹²    moles/litre or less and more preferably 10⁻⁸ to 10⁻¹² moles/litre.-   Aspect D-4: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, and/or D-1 to D-3 that can specifically bind to    multiscavenger receptors with a rate of association (k_(on)-rate) of    between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷    M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect D-5: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, and/or D-1 to D-4 that can specifically bind to    multiscavenger receptors with a rate of dissociation (k_(off) rate)    between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹,    more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴    s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect D-6: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, and/or D-1 to D-5 that can specifically bind to    multiscavenger receptors with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM.    The amino acid sequences according to aspects D-1 to D-6 may in    particular be an amino acid sequence according to any of the aspects    A-1 to A-22.-   Aspect E-1: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7 and/or D1 to D-6, that is a naturally occurring    amino acid sequence (from any suitable species) or a synthetic or    semi-synthetic amino acid sequence.-   Aspect E-2: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, D1 to D-6, and/or E-1 that comprises an    immunoglobulin fold or that under suitable conditions is capable of    forming an immunoglobulin fold.-   Aspect E-3: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, D1 to D-6, and/or D-1 or D-2, that is an    immunoglobulin sequence.-   Aspect E-4: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-3, that is a    naturally occurring immunoglobulin sequence (from any suitable    species) or a synthetic or semi-synthetic immunoglobulin sequence.-   Aspect E-5: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-4 that is a humanized    immunoglobulin sequence, a camelized immunoglobulin sequence or an    immunoglobulin sequence that has been obtained by techniques such as    affinity maturation.-   Aspect E-6: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-5 that essentially    consists of a light chain variable domain sequence (e.g. a    V_(L)-sequence); or of a heavy chain variable domain sequence (e.g.    a V_(H)-sequence).-   Aspect E-7: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-6, that essentially    consists of a heavy chain variable domain sequence that is derived    from a conventional four-chain antibody or that essentially consist    of a heavy chain variable domain sequence that is derived from heavy    chain antibody.-   Aspect E-8: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-7, that essentially    consists of a domain antibody (or an An amino acid sequence that is    suitable for use as a domain antibody), of a single domain antibody    (or an An amino acid sequence that is suitable for use as a single    domain antibody), of a “dAb” (or an An amino acid sequence that is    suitable for use as a dAb) or of a Nanobody (including but not    limited to a V_(TI) sequence).-   Aspect E-9: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-8 that essentially    consists of a Nanobody.-   Aspect E-10: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-9 that essentially    consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 1 to 22, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect E-11: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-10, that essentially    consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        An amino acid sequences of SEQ ID NO's: 308-333, in which for        the purposes of determining the degree of amino acid identity,        the amino acid residues that form the CDR sequences are        disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect E-12: An amino acid sequence according to any of aspects B-1    to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-11 that essentially    consists of a humanized Nanobody.-   Aspect E-13: An amino acid sequence according to any of the aspects    B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-11, that in    addition to the at least one binding site for binding formed by the    CDR sequences, contains one or more further binding sites for    binding against other antigens, proteins or targets.    The amino acid sequences according to aspects E-1 to E-13 may in    particular be an amino acid sequence according to any of the aspects    A-1 to A-22.-   Aspect F-1: An amino acid sequence that essentially consists of 4    framework regions (FR1 to FR4, respectively) and 3 complementarity    determining, regions (CDR1 to CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 152-177;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 152-177;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 152-177;    -   and/or    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 204-229;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 204-229;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 204-229;    -   and/or    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 256-281;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's:    256-281;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's:    256-281.    Such an amino acid sequence is preferably directed against    multiscavenger receptors and/or an amino acid sequence that can    specifically bind to multiscavenger receptors. Also, such an amino    acid sequence is preferably an amino acid sequence according to any    of the aspects A-1 to A-22, C-1 to C-7. D1 to D-6 and/or E-1 to    E-13.-   Aspect F-2: An amino acid sequence that essentially consists of 4    framework regions (FR1 to FR4, respectively) and 3 complementarity    determining regions (CDR1 to CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 152-177;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 152-177;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 152-177;    -   and    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 204-229;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 204-229;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 204-229;    -   and    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 256-281;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 256-281;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 256-281.        Such an amino acid sequence is preferably directed against        multiscavenger receptors and/or an amino acid sequence that can        specifically bind to multiscavenger receptors. Also, such an        amino acid sequence is preferably an amino acid sequence        according to any of the aspects A-1 to A-22, C-1 to C-7, D1 to        D-6 and/or E-1 to E-13.-   Aspect F-3: An amino acid sequence according to any of aspects F-1    and F-2, in which the CDR sequences of said amino acid sequence have    at least 70% amino acid identity, preferably at least 80% amino acid    identity, more preferably at least 90% amino acid identity, such as    95% amino acid identity or more or even essentially 100% amino acid    identity with the CDR sequences of at least one of the amino acid    sequences of SEQ ID NO's: 308-333.    Such an amino acid sequence is preferably directed against    multiscavenger receptors and/or an amino acid sequence that can    specifically bind to multiscavenger receptors. Also, such an amino    acid sequence is preferably an amino acid sequence according to any    of the aspects A-1 to A-22, C-1 to C-7, D1 to D-6 and/or E-1 to    E-13.-   Aspect F-4: An amino acid sequence according to any of aspects F-1    to F-3 that is directed against multiscavenger receptors and that    cross-blocks the binding of at least one of the amino acid sequences    according to any of aspects the amino acid sequences of SEQ ID NO's:    308-333.-   Aspect F-5: An amino acid sequence according to any of aspects F-1    to F-3 that is directed against multiscavenger receptors and that is    cross-blocked from binding to multiscavenger receptors by at least    one of the amino acid sequences of SEQ ID NO's: 308-333.-   Aspect F-6: Amino acid sequence according to any of aspects F-4 or    F-5 wherein the ability of said amino acid sequence to cross-block    or to be cross-blocked is detected in a Biacore assay.-   Aspect F-7: Amino acid sequence according to any of aspects F4 or    F-5 wherein the ability of said amino acid sequence to cross-block    or to be cross-blocked is detected in an ELISA assay.-   Aspect F-8: An amino acid sequence according to any of aspects F-1    to F-7, that is in essentially isolated form.-   Aspect F-9: An amino acid sequence according to any of aspects F-1    to F-8, for administration to a subject, wherein said an amino acid    sequence does not naturally occur in said subject.-   Aspect F-10: An amino acid sequence according to any of aspects F-1    to F-9, that can specifically bind to multiscavenger receptors with    a dissociation constant (K_(D)) of 10⁻⁵ to 10¹² moles/litre or less,    and preferably 10⁻⁷ to 10¹² moles/litre or less and more preferably    10⁸ to 10⁻¹² moles/litre.-   Aspect F-11: An amino acid sequence according to any of aspects F-1    to F-10, that can specifically bind to multiscavenger receptors with    a rate of association rate) of between 10² M⁻¹s⁻¹ to about 10⁷    M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more    preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect F-12: An amino acid sequence according to any of aspects F-1    to F-11, that can specifically bind to multiscavenger receptors with    a rate of dissociation (k_(off) rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹    preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between    10³ and 10⁻⁶ such as between 10¹ s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect F-13: An amino acid sequence according to any of aspects F-1    to F-12, that can specifically bind to multiscavenger receptors with    an affinity less than 500 nM, preferably less than 200 nM, more    preferably less than 10 nM, such as less than 500 pM.-   Aspect F-14: An amino acid sequence according to any of aspects F-1    to F-13, that is a naturally occurring amino acid sequence (from any    suitable species) or a synthetic or semi-synthetic amino acid    sequence.-   Aspect F-15: An amino acid sequence according to any of aspects F-1    to F-14, that comprises an immunoglobulin fold or that under    suitable conditions is capable of forming an immunoglobulin fold.-   Aspect F-16: An amino acid sequence according to any of aspects F-1    to F-15, that is an immunoglobulin sequence.-   Aspect F-17: An amino acid sequence according to any of aspects F-1    to F-16, that is a naturally occurring immunoglobulin sequence (from    any suitable species) or a synthetic or semi-synthetic    immunoglobulin sequence.-   Aspect F-18: An amino acid sequence according to any of aspects F-1    to F-17, that is a humanized immunoglobulin sequence, a camelized    immunoglobulin sequence or an immunoglobulin sequence that has been    obtained by techniques such as affinity maturation.-   Aspect F-19: An amino acid sequence according to any of aspects F-1    to F-19, that essentially consists of a light chain variable domain    sequence (e.g. a V_(L)-sequence); or of a heavy chain variable    domain sequence (e.g. a V_(H)-sequence).-   Aspect F-20: An amino acid sequence according to any of aspects F-1    to F-19, that essentially consists of a heavy chain variable domain    sequence that is derived from, a conventional four-chain antibody or    that essentially consist of a heavy chain variable domain sequence    that is derived from heavy chain antibody.-   Aspect F-21: An amino acid sequence according to any of aspects F-1    to F-20, that essentially consists of a domain antibody (or an amino    acid sequence that is suitable for use as a domain antibody), of a    single domain antibody (or an amino acid sequence that is suitable    for use as a single domain antibody), of a “dAb” (or an amino acid    sequence that is suitable for use as a dAb) or of a Nanobody    (including but not limited to a V_(HH) sequence).-   Aspect F-22: An amino acid sequence according to any of aspects F-1    to F-21, that essentially consists of a Nanobody.-   Aspect F-23: An amino acid sequence according to any of aspects F-1    to F-22, that essentially consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 1 to 22, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect F-24: An amino acid sequence according to any of aspects F-1    to F-23, that essentially consists of a Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        amino acid sequences of SEQ ID NO's: 308-333, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect F-25: An amino acid sequence according to any of aspects F-1    to F-24, that essentially consists of a humanized Nanobody.-   Aspect G-1: An amino acid sequence according to any of the preceding    aspects, that in addition to the at least one binding site for    binding formed by the CDR sequences, contains one or more further    binding sites for binding against another antigen, protein or    target.-   Aspect H-1: Nanobody that is directed against and/or that can    specifically bind to multiscavenger receptors.-   Aspect H-2: Nanobody according to aspect H-1, that is in essentially    isolated form.-   Aspect H-3: Nanobody according to any of aspects H-1 to H-2, that    can specifically bind to multiscavenger receptors with a    dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/litre or less,    and preferably 10⁻⁷ to 10⁻¹² moles/litre or less and more preferably    10⁻⁸ to 10⁻¹² moles/litre.-   Aspect H-4: Nanobody according to any of aspects H-1 to H-3, that    can specifically bind to multiscavenger receptors with a rate of    association (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹,    preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably    between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and    10⁷ M⁻¹s⁻¹.-   Aspect H-5: Nanobody according to any of aspects H-1 to H-4, that    can specifically bind to multiscavenger receptors with a rate of    dissociation (k_(off) rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably    between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and    10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect H-6: Nanobody according to any of aspects H-1 to H-5, that    can specifically bind to multiscavenger receptors with an affinity    less than 500 nM, preferably less than 200 nM, more preferably less    than 10 nM, such as less than 500 pM.-   Aspect H-7: Nanobody according to any of aspects H-1 to H-6, that is    a naturally occurring Nanobody (from any suitable species) or a    synthetic or semi-synthetic Nanobody.-   Aspect H-8: Nanobody according to any of aspects to H-1 to H-7, that    is a V_(HH) sequence, a partially humanized V_(HH) sequence, a fully    humanized V_(HH) sequence, a camelized heavy chain variable domain    or a Nanobody that has been obtained by techniques such as affinity    maturation.-   Aspect H-9: Nanobody according to any of aspects H-1 to H-8, that    -   i) has at least 80% amino acid identity with at least one of the        An amino acid sequences of SEQ ID NO's: 1 to 22, in which for        the purposes of determining the degree of amino acid identity,        the amino acid residues that form the CDR sequences are        disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect H-10: Nanobody according to any of aspects H-1 to H-9, that    -   i) has at least 80% amino acid identity with at least one of the        An amino acid sequences of SEQ ID NO's: 308-333, in which for        the purposes of determining the degree of amino acid identity,        the amino acid residues that form the CDR sequences are        disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect H-11: Nanobody according to any of aspects H-1 to H-10, in    which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 152-177;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 152-177;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 152-177;    -   and/or    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 204-229;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 204-229;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 204-229;    -   and/or    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 256-281;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 256-281;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 256-281.-   Aspect H-12: Nanobody according to any of aspects H-1 to H-11, in    which:    -   CDR1 is chosen from the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 152-177;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 152-177;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 152-177;    -   and    -   CDR2 is chosen from the group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 204-229;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 204-229;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 204-229;    -   and    -   CDR3 is chosen from the group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 256-281;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 256-281;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 256-281.-   Aspect H-13: Nanobody according to any of aspects H-1 to H-12, in    which the CDR sequences have at least 70% amino acid identity,    preferably at least 80% amino acid identity, more preferably at    least 90% amino acid identity, such as 95% amino acid identity or    more or even essentially 100% amino acid identity with the CDR    sequences of at least one of the amino acid sequences of SEQ ID    NO's: 308-333.-   Aspect H-14: Nanobody according to any of aspects H-1 to H-13, which    is a partially humanized Nanobody.-   Aspect H-15: Nanobody according to any of aspects H-1 to H-14, which    is a fully humanized Nanobody.-   Aspect H-16: Nanobody according to any of aspects H-1 to H-15, that    is chosen from the group consisting of SEQ ID NO's: 308-333 or from    the group consisting of from amino acid sequences that have more    than 80%, preferably more than 90%, more preferably more than 95%,    such as 99% or more sequence identity (as defined herein) with at    least one of the amino acid sequences of SEQ ID NO's: 308-333.-   Aspect HH-17: Nanobody according to any of aspects H-1 to H-16,    which is a humanized Nanobody that is chosen from the group    consisting of humanized Nanobody sequences or from the group    consisting of from amino acid sequences that have more than 80%,    preferably more than 90%, more preferably more than 95%, such as 99%    or more sequence identity (as defined herein) with at least one of    the amino acid sequences of humanized Nanobody sequences.-   Aspect H-18: Nanobody according to any of aspects H-1 to H-17, that    is chosen from the group consisting of SEQ ID NO's: 308-333 or from    the group consisting of humanized Nanobody sequences.-   Aspect H-19: Nanobody directed against multiscavenger receptors that    cross-blocks the binding of at least one of the amino acid sequences    of SEQ ID NO's: 308-333 to multiscavenger receptors.-   Aspect H-20: Nanobody directed against multiscavenger receptors that    is cross-blocked from binding to multiscavenger receptors by at    least one of the amino acid sequences of SEQ ID NO's: 308-333.-   Aspect H-21: Nanobody according to any of aspects H-19 or H-20    wherein the ability of said Nanobody to cross-block or to be    cross-blocked is detected in a Biacore assay.-   Aspect H-22: Nanobody according to any of aspects H-19 to H-21    wherein the ability of said Nanobody to cross-block or to be    cross-blocked is detected in an ELISA assay.-   Aspect K-1: Polypeptide that comprises or essentially consists of    one or more amino acid sequences according to any of aspects A-1 to    A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25    or (3-1 and/or one or more Nanobodies according to any of aspects    H-1 to H-22, and optionally further comprises one or more peptidic    linkers.-   Aspect K-2: Polypeptide according to aspect K-1, in which said one    or more binding units are immunoglobulin sequences.-   Aspect K-3: Polypeptide according to any of aspects K-1 or K-2, in    which said one or more other groups, residues, moieties or binding    units are chosen from the group consisting of domain antibodies,    amino acid sequences that are suitable for use as a domain antibody,    single domain antibodies, amino acid sequences that are suitable for    use as a single domain antibody, “dAb”'s, amino acid sequences that    are suitable for use as a dAb, or Nanobodies.-   Aspect K-4: Polypeptide according to any of aspects K-1 to K-3, in    which said one or more amino acid sequences of the invention are    immunoglobulin sequences.-   Aspect K-5: Polypeptide according to any of aspects K-1 to K-4, in    which said one or more amino acid sequences of the invention are    chosen from the group consisting of domain antibodies, amino acid    sequences that are suitable for use as a domain antibody, single    domain antibodies, amino acid sequences that are suitable for use as    a single domain antibody, “dAb”'s, amino acid sequences that are    suitable for use as a dAb, or Nanobodies.-   Aspect K-6: Polypeptide according to any of aspects K-1 to K-5, that    comprises or essentially consists of one or more Nanobodies    according to any of aspects H-1 to H-22 and in which said one or    more other binding units are Nanobodies.-   Aspect K-7: Polypeptide according to any of aspects K-1 to K-6,    wherein at least one binding unit is a multivalent construct.-   Aspect K-8: Polypeptide according to any of aspects K-1 to K-8,    wherein at least one binding unit is a multiparatopic construct.-   Aspect K-9: Polypeptide according to any of aspects K-1 to K-8,    wherein at least one binding unit is a multispecific construct.-   Aspect K-10: Polypeptide according to any of aspects K-1 to K-9,    which has an increased half-life, compared to the corresponding    amino acid sequence according to any of aspects A-1 to A-22, B-1 to    B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1 per se    or Nanobody according to any of aspects H-1 to H-22 per se,    respectively.-   Aspect K-11: Polypeptide according to aspect K-10, in which said one    or more other binding units provide the polypeptide with increased    half-life, compared to the corresponding amino acid sequence    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1 per se or Nanobody according    to any of aspects H-1 to H-22 per se, respectively.-   Aspect K-12: Polypeptide according to aspect K-10 or K-11, in which    said one or more other binding units that provide the polypeptide    with increased half-life is chosen from the group consisting of    serum proteins or fragments thereof, binding units that can bind to    serum proteins, an Fc portion, and small proteins or peptides that    can bind to serum proteins.-   Aspect K-13: Polypeptide according to any of aspects K-10 to K-12,    in which said one or more other binding units that provide the    polypeptide with increased half-life is chosen from the group    consisting of human serum albumin or fragments thereof.-   Aspect K-14: Polypeptide according to any of aspect K-10 to K-13, in    which said one or more other binding units that provides the    polypeptide with increased half-life are chosen from the group    consisting of binding units that can bind to serum albumin (such as    human serum albumin) or a serum immunoglobulin (such as IgG).-   Aspect K-15: Polypeptide according to any of aspects K-10 to K-14,    in which said one or more other binding units that provides the    polypeptide with increased half-life are chosen from the group    consisting of domain antibodies, amino acid sequences that are    suitable for use as a domain antibody, single domain antibodies,    amino acid sequences that are suitable for use as a single domain    antibody, “dAb”'s, amino acid sequences that are suitable for use as    a dAb, or Nanobodies that can bind to serum albumin (such as human    serum albumin) or a serum immunoglobulin (such as IgG).-   Aspect K-16: Polypeptide according to aspect K-10 to K-15, in which    said one or more other binding units that provides the polypeptide    with increased half-life is a Nanobody that can bind to serum    albumin (such as human serum albumin) or a serum immunoglobulin    (such as IgG).-   Aspect K-17: Polypeptide according to any of aspects K-10 to K-16,    that has a serum half-life that is at least 1.5 times, preferably at    least 2 times, such as at least 5 times, for example at least 10    times or more than 20 times, greater than the half-life of the    corresponding amino acid sequence according to any of aspects A-1 to    A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25    or G-1 per se or Nanobody according to any of aspects H-1 to H-22    per se, respectively.-   Aspect K-18: Polypeptide according to any of aspects K-10 to K-17,    that has a serum half-life that is increased with more than 1 hours,    preferably more than 2 hours, more preferably more than 6 hours,    such as more than 12 hours, or even more than 24, 48 or 72 hours,    compared to the corresponding amino acid sequence according to any    of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4. D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1 per se or Nanobody according to any of    aspects H-1 to H-22 per se, respectively.-   Aspect K-19: Polypeptide according to any of aspects K-1 to K-18,    that has a serum half-life in human of at least about 12 hours,    preferably at least 24 hours, more preferably at least 48 hours,    even more preferably at least 72 hours or more; for example, of at    least 5 days (such as about 5 to 10 days), preferably at least 9    days (such as about 9 to 14 days), more preferably at least about 10    days (such as about 10 to 15 days), or at least about 11 days (such    as about 11 to 16 days), more preferably at least about 12 days    (such as about 12 to 18 days or more), or more than 14 days (such as    about 14 to 19 days).-   Aspect L-1: Compound or construct, that comprises or essentially    consists of one or more amino acid sequences according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1 and/or one or more Nanobodies according to    any of aspects H-1 to H-22, and optionally further comprises one or    more other groups, residues, moieties or binding units, optionally    linked via one or more linkers.-   Aspect L-2: Compound or construct according to aspects L-1, in which    said one or more other groups, residues, moieties or binding units    are amino acid sequences.-   Aspect L-3: Compound or construct according to aspect L-1 or L-2, in    which said one or more linkers, if present, are one or more amino    acid sequences.-   Aspect L-4: Compound or construct according to any of aspects L-1 to    L-3, in Which said one or more other groups, residues, moieties or    binding units are immunoglobulin sequences.-   Aspect L-5: Compound or construct according to any of aspects L-1 to    L-4, in which said one or more other groups, residues, moieties or    binding units are chosen from the group consisting of domain    antibodies, amino acid sequences that are suitable for use as a    domain antibody, single domain antibodies, amino acid sequences that    are suitable for use as a single domain antibody, “dAb”'s, amino    acid sequences that are suitable for use as a dAb, or Nanobodies.-   Aspect L-6: Compound or construct according to any of aspects L-1 to    L-5, in which said one or more amino acid sequences of the invention    are immunoglobulin sequences.-   Aspect L-7: Compound or construct according to any of aspects L-1 to    L-6, in which said one or more amino acid sequences of the invention    are chosen from the group consisting of domain antibodies, amino    acid sequences that are suitable for use as a domain antibody,    single domain antibodies, amino acid sequences that are suitable for    use as a single domain antibody, “dAb”'s, amino acid sequences that    are suitable for use as a dAb, or Nanobodies.-   Aspect L-8: Compound or construct, that comprises or essentially    consists of one or more Nanobodies according to any of aspects H-1    to H-22 and in which said one or more other groups, residues,    moieties or binding units are Nanobodies.-   Aspect L-9: Compound or construct according to any of aspects L-1 to    L-9, which is a multivalent construct.-   Aspect L-10: Compound or construct according to any of aspects L-1    to L-10, which is a multispecific construct.-   Aspect L-11: Compound or construct according to any of aspects L-1    to L-10, which has an increased half-life, compared to the    corresponding amino acid sequence according to any of aspects A-1 to    A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25    or G-1 per se or Nanobody according to any of aspects H-1 to H-22    per se, respectively.-   Aspect L-12: Compound or construct according to aspect L-1 to L-11,    in which said one or more other groups, residues, moieties or    binding units provide the compound or construct with increased    half-life, compared to the corresponding amino acid sequence    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1 per se or Nanobody according    to any of aspects H-1 to H-22 per se, respectively.-   Aspect L-13: Compound or construct according to aspect L-12, in    which said one or more other groups, residues, moieties or binding    units that provide the compound or construct with increased    half-life is chosen from the group consisting of serum proteins or    fragments thereof, binding units that can bind to serum proteins, an    Fc portion, and small proteins or peptides that can bind to serum    proteins.-   Aspect L-14: Compound or construct according to aspect L-12 or L-13,    in which said one or more other groups, residues, moieties or    binding units that provide the compound or construct with increased    half-life is chosen from the group consisting of human serum    albumin, or fragments thereof,-   Aspect L-15: Compound or construct according to any of aspects L-12    to L-14, in which said one or more other groups, residues, moieties    or binding units that provides the compound or construct with    increased half-life are chosen from the group consisting of binding    units that can bind to serum albumin (such as human serum albumin)    or a serum immunoglobulin (such as IgG).-   Aspect L-16: Compound or construct according to any of aspects L-12    to L-14, in which said one or more other groups, residues, moieties    or binding units that provides the compound or construct with    increased half-life are chosen from the group consisting of domain    antibodies, amino acid sequences that are suitable for use as a    domain antibody, single domain antibodies, amino acid sequences that    are suitable for use as a single domain antibody, “dAb”'s, amino    acid sequences that are suitable for use as a dAb, or Nanobodies    that can bind to serum albumin (such as human serum albumin) or a    serum immunoglobulin (such as IgG).-   Aspect L-17: Compound or construct according to any of aspects L-12    to L-14, in which said one or more other groups, residues, moieties    or binding units that provides the compound or construct with    increased half-life is a Nanobody that can bind to serum albumin    (such as human serum albumin) or a serum immunoglobulin (such as    IgG).-   Aspect L-18: Compound or construct according to any of aspects L-12    to L-17, that has a serum half-life that is at least 1.5 times,    preferably at least 2 times, such as at least 5 times, for example    at least 10 times or more than 20 times, greater than the half-life    of the corresponding amino acid sequence according to any of aspects    A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to    F-25 or G-1 per se or Nanobody according to any of aspects H-1 to    H-22 per se, respectively.-   Aspect L-19: Compound or construct according to any of aspects L-12    to L-18, that has a serum half-life that is increased with more than    1 hours, preferably more than 2 hours, more preferably more than 6    hours, such as more than 12 hours, or even more than 24, 48 or 72    hours, compared to the corresponding amino acid sequence according    to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6,    E-1 to E-13, F-1 to F-25 or G-1 per se or Nanobody according to any    of aspects H-1 to H-22 per se, respectively.-   Aspect L-20: Compound or construct according to any of aspects L-12    to L-19, that has a serum half-life in human of at least about 12    hours, preferably at least 24 hours, more preferably at least 48    hours, even more preferably at least 72 hours or more; for example,    of at least 5 days (such as about 5 to 10 days), preferably at least    9 days (such as about 9 to 14 days), more preferably at least about    10 days (such as about 10 to 15 days), or at least about 11 days    (such as about 11 to 16 days), more preferably at least about 12    days (such as about 12 to 18 days or more), or more than 14 days    (such as about 14 to 19 days).-   Aspect L-21: Monovalent construct, comprising or essentially    consisting of one amino acid sequence according to any of aspects    A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13. F-1 to    F-25 or G-1 and/or one Nanobody according to any of aspects H-1 to    H-22.-   Aspect L-22: Monovalent construct according to aspect L-21, in which    said amino acid sequence of the invention is chosen from the group    consisting of domain antibodies, amino acid sequences that are    suitable for use as a domain antibody, single domain antibodies,    amino acid sequences that are suitable for use as a single domain    antibody, “dAb”'s, amino acid sequences that are suitable for use as    a dAb, or Nanobodies.-   Aspect L-23: Monovalent construct, comprising or essentially    consisting of one Nanobody according to any of aspects H-1 to H-22.-   Aspect M-1: Nucleic acid or nucleotide sequence, that encodes an    amino acid sequence according to any of aspects A-1 to A-22, B-1 to    B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, a    Nanobody according to any of aspects H-1 to H-22, a compound or    construct according to any of aspects that is such that it can be    obtained by expression of a nucleic acid or nucleotide sequence    encoding the same, or a monovalent construct according to any of    aspects-   Aspect M-2: Nucleic acid or nucleotide sequence according to aspect    that is in the form of a genetic construct.-   Aspect N-1: Host or host cell that expresses, or that under suitable    circumstances is capable of expressing, an amino acid sequence    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1, a Nanobody according to any    of aspects H-1 to H-22, a polypeptide according to any of aspects    K-1 to K-19, a compound or construct according to any of aspects L-1    to L-21 that is such that it can be obtained by expression of a    nucleic acid or nucleotide sequence encoding the same, or a    monovalent construct according to any of aspects L-22 or L-23;    and/or that comprises a nucleic acid or nucleotide sequence    according to aspect M-1 or a genetic construct according to aspect    M-2.-   Aspect O-1: Composition comprising at least one amino acid sequence    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1, Nanobody according to any    of aspects H-1 to H-22, polypeptide according to any of aspects K-1    to K-19, compound or construct according to any of aspects L-1 to    L-21, monovalent construct according to any of aspects L-22 or L-23,    or nucleic acid or nucleotide sequence according to aspects M-1 or    M-2.-   Aspect O-2: Composition according to aspect O-1, which is a    pharmaceutical composition.-   Aspect O-3: Composition according to aspect O-2, which is a    pharmaceutical composition, that further comprises at least one    pharmaceutically acceptable carrier, diluent or excipient and/or    adjuvant, and that optionally comprises one or more further    pharmaceutically active polypeptides and/or compounds.-   Aspect P-1: Method for producing an amino acid sequence according to    any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1    to E-13, F-1 to F-25 or G-1, a Nanobody according to any of aspects    H-1 to H-22, a polypeptide according to any of aspects K-1 to K-19,    a compound or construct according to any of aspects L-1 to L-21 that    is such that it can be obtained by expression of a nucleic acid or    nucleotide sequence encoding the same, or a monovalent construct    according to any of aspects L-22 or L-23, said method at least    comprising the steps of:    -   a) expressing, in a suitable host cell or host organism or in        another suitable expression system, a nucleic acid or nucleotide        sequence according to aspect M-1, or a genetic construct        according to aspect M-2;    -   optionally followed by:    -   b) isolating and/or purifying the amino acid sequence according        to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to        D-6, E-1 to E-13, F-1 to F-25 or G-1, a Nanobody according to        any of aspects H-1 to H-22, a polypeptide according to any of        aspects K-1 to K-19, a compound or construct according to any of        aspects L-1 to L-21, or a monovalent construct according to any        of aspects L-22 or L-23 thus obtained.-   Aspect P-2: Method for producing an amino acid sequence according to    any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1    to E-13, F-1 to F-25 or GA, a Nanobody according to any of aspects    H-1 to H-22, a polypeptide according to any of aspects K-1 to K-19,    a compound or construct according to any of aspects L-1 to L-21 that    is such that it can be obtained by expression of a nucleic acid or    nucleotide sequence encoding the same, or a monovalent construct    according to any of aspects L-22 or L-23, said method at least    comprising the steps of:    -   a) cultivating and/or maintaining a host or host cell according        to aspect . . . under conditions that are such that said host or        host cell expresses and/or produces at least one amino acid        sequence according to any of aspects A-1 to A-22, B-1 to B-7,        C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1,        Nanobody according to any of aspects H-1 to H-22, a polypeptide        according to any of aspects K-1 to K-19, a compound or construct        according to any of aspects L-1 to L-21, or monovalent construct        according to any of aspects L-22 or L-23;    -   optionally followed by:    -   b) isolating and/or purifying the amino acid sequence according        to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to        D-6, E-1 to E-13, F-1 to F-25 or G-1, Nanobody according to any        of aspects H-1 to H-22, a polypeptide according to any of        aspects K-1 to K-19, a compound or construct according to any of        aspects L-1 to L-21, or monovalent construct according to any of        aspects L-22 or L-23 thus obtained.-   Aspect Q-1: Method for screening amino acid sequences directed    against multiscavenger receptors that comprises at least the steps    of:    -   a) providing a set, collection or library of nucleic acid        sequences encoding amino acid sequences;    -   b) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode an amino acid        sequence that can bind to and/or has affinity for multiscavenger        receptors and that is cross-blocked or is cross blocking a        Nanobody of the invention, e.g. SEQ ID NO: 308-333 (Table-1);        and    -   c) isolating said nucleic acid sequence, followed by expressing        said amino acid sequence.-   Aspect R-1: Method for the prevention and/or treatment of at least    one a disease wherein multiscanvenger receptors are implicated, said    method comprising administering, to a subject in need thereof, a    pharmaceutically active amount of at least one amino acid sequence    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4. D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1, Nanobody according to any    of aspects H-1 to H-22, polypeptide according to any of aspects K-1    to K-19, compound or construct according to any of aspects L-1 to    L-21, monovalent construct according to any of aspects L-22 or L-23;    or composition according to aspect O-2 or O-3.-   Aspect R-2: Method for the prevention and/or treatment of at least    one disease or disorder that is associated with multiscavenger    receptors, with its biological or pharmacological activity, and/or    with the biological pathways or signalling in which multiscavenger    receptors is involved, said method comprising administering, to a    subject in need thereof, a pharmaceutically active amount of at    least one amino acid sequence according to any of aspects A-1 to    A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25    or O-1, Nanobody according to any of aspects H-1 to H-22,    polypeptide according to any of aspects K-1 to K-19, compound or    construct according to any of aspects L-1 to L-21, monovalent    construct according to any of aspects L-22 or L-23; or composition    according to aspect O-2 or O-3.-   Aspect R-3: Method for the prevention and/or treatment of at least    one disease or disorder that can be prevented and/or treated by    administering, to a subject in need thereof, at least one amino acid    sequence according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to    C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, Nanobody according    to any of aspects H-1 to H-22, polypeptide according to any of    aspects K-1 to K-19, compound or construct according to any of    aspects L-1 to L-21, monovalent construct according to any of    aspects L-22 or L-23; or composition according to aspect O-2 or O-3,    said method comprising administering, to a subject in need thereof,    a pharmaceutically active amount of at least one at least one amino    acid sequence according to any of aspects A-1 to A-22, B-1 to B-7,    C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, Nanobody    according to any of aspects H-1 to H-22, polypeptide according to    any of aspects K-1 to K-19, compound or construct according to any    of aspects L-1 to L-21, monovalent construct according to any of    aspects L-22 or L-23; or composition according to aspect O-2 or O-3.-   Aspect R-4: Method for immunotherapy, said method comprising    administering, to a subject in need thereof, a pharmaceutically    active amount of at least one amino acid sequence according to any    of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1, Nanobody according to any of aspects H-1    to H-22, polypeptide according to any of aspects K-1 to K-19,    compound or construct according to any of aspects L-1 to L-21,    monovalent construct according to any of aspects L-22 or L-23; or    composition according to aspect O-2 or O-3.-   Aspect R-5: Use of an amino acid sequence according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1, a Nanobody according to any of aspects H-1    to H-22, a polypeptide according to any of aspects K-1 to K-19, a    compound or construct according to any of aspects L-1 to L-21, or a    monovalent construct according to any of aspects L-22 or L-23 in the    preparation of a pharmaceutical composition for prevention and/or    treatment of at least one a disease wherein multiscanvenger    receptors are implicated; and/or for use in one or more of the    methods according to aspects R-1 to R-3.-   Aspect R-6: Amino acid sequence according to any of aspects A-1 to    A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25    or G-1, Nanobody according to any of aspects H-1 to H-22,    polypeptide according to any of aspects K-1 to K-19, compound or    construct according to any of aspects L-1 to L-21, monovalent    construct according to any of aspects L-22 or L-23; or composition    according to aspect O-2 or O-3 for the prevention and/or treatment    of at least one a disease wherein multiscanvenger receptors are    implicated.-   Aspect S-1: Part or fragment of an amino acid sequence according to    any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1    to E-13, F-1 to F-25 or G-1, or of a Nanobody according to any of    aspects H-1 to H-22.-   Aspect S-2: Part or fragment according to aspect S-1, that can    specifically bind to multiscavenger receptors.-   Aspect S-3: Part of fragment according to any of aspects S-1 or S-2,    that can specifically bind to multiscavenger receptors with a    dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/litre or less,    and preferably 10⁻⁷ to 10⁻¹² moles/litre or less and more preferably    10⁻⁸ to 10⁻¹² moles/litre.-   Aspect 5-4: Part or fragment according to any of aspects S-1 to S-3,    that can specifically bind to multiscavenger receptors with a rate    of association (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷    M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more    preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect 5-5: Part or fragment according to any of aspects S-1 to S-4,    that can specifically bind to multiscavenger receptors with a rate    of dissociation (k_(off) rate) between s⁻¹ and 10⁻⁶ s⁻¹ preferably    between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and    10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect S-6: Compound or construct, that comprises or essentially    consists of one or more parts or fragments according to any of    aspects S-1 to S-4, and optionally further comprises one or more    other groups, residues, moieties or binding units, optionally linked    via one or more linkers.-   Aspect S-7: Compound or construct according to aspect S-6, in which    said one or more other groups, residues, moieties or binding units    are amino acid sequences.-   Aspect S-8: Compound or construct according to aspect S-6, or S-7,    in which said one or more linkers, if present, are one or more amino    acid sequences.-   Aspect S-9: Nucleic acid or nucleotide sequence, that encodes a part    or fragment according to any of aspects S-1 to S-7 or a compound or    construct according to aspect 5-8.-   Aspect S-10: Composition, comprising at least one part or fragment    according to any of aspects S-1 to S-7, compound or construct    according to any of aspects S-6 to S-8, or nucleic acid or    nucleotide sequence according to aspect S-9.-   Aspect T-1: Derivative of an amino acid sequence according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or O-1, or of a Nanobody according to any of    aspects H-1 to H-22.-   Aspect T-2: Derivative according to aspect T-1, that can    specifically bind to multiscavenger receptors.-   Aspect T-3: Derivative according to any of aspects T-1 or T-2, that    can specifically bind to multiscavenger receptors with a    dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/litre or less,    and preferably 10⁻⁷ to 10⁻¹² moles/litre or less and more preferably    10⁻⁸ to 10⁻¹² moles/litre.-   Aspect T-4: Derivative according to any of aspects T-1 to T-3, that    can specifically bind to multiscavenger receptors with a rate of    association (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹,    preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably    between 10⁴ M⁻¹5⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and    10⁷ M⁻¹s⁻¹.-   Aspect T-5: Derivative according to any of aspects T-1 to T-4, that    can specifically bind to multiscavenger receptors with a rate of    dissociation (k_(off) rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably    between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and    10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect T-6: Derivative of a polypeptide according to any of aspects    K-1 to K-19 or compound or construct according to any of aspects L-1    to L-23.-   Aspect T-7: Derivative according to aspect T-6, that can    specifically bind to multiscavenger receptors.-   Aspect T-8: Derivative according to any of aspects T-6 or T-7, that    can specifically bind to multiscavenger receptors with a    dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less,    and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably    10⁻⁸ to 10⁻¹² moles/liter.-   Aspect T-9: Derivative according to any of aspects T-6 to T-8, that    can specifically bind to multiscavenger receptors with a rate of    association (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹,    preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably    between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and    10⁷ M⁻¹s⁻¹.-   Aspect T-10: Derivative according to any of aspects T-6 to T-9, that    can specifically bind to multiscavenger receptors with a rate of    dissociation (k_(off) rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably    between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and    10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect T-11: Derivative according to any of aspects T-1 to T-10,    that has a serum half-life that is at least 1.5 times, preferably at    least 2 times, such as at least 5 times, for example at least 10    times or more than 20 times, greater than the half-life of the    corresponding amino acid sequence according to any of aspects A-1 to    A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25    or G-1 per se, Nanobody according to any of aspects H-1 to H-22 per    se, polypeptide according to any of aspects K-1 to K-19 or compound    or construct according to any of aspects L-1 to L-23 per se.-   Aspect T-12: Derivative according to any of aspects T-1 to T-11,    that has a serum half-life that is increased with more than 1 hours,    preferably more than 2 hours, more preferably more than 6 hours,    such as more than 12 hours, or even more than 24, 48 or 72 hours,    compared to the corresponding amino acid sequence according to any    of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1 per se, Nanobody according to any of    aspects H-1 to H-23 per se, polypeptide according to any of aspects    K-1 to K-19 or compound or construct according to any of aspects L-1    to L-23 per se, respectively.-   Aspect T-13: Derivative according to any of aspects T-1 to T-12,    that has a serum half-life in human of at least about 12 hours,    preferably at least 24 hours, more preferably at least 48 hours,    even more preferably at least 72 hours or more; for example, at    least 5 days (such as about 5 to 10 days), preferably at least 9    days (such as about 9 to 14 days), more preferably at least about 10    days (such as about 10 to 15 days), or at least about 11 days (such    as about 11 to 16 days), more preferably at least about 12 days    (such as about 12 to 18 days or more), or more than 14 days (such as    about 14 to 19 days).-   Aspect T-14: Derivative according to any of aspects T-1 to T-13,    that is a pegylated derivative.-   Aspect T-15: Compound or construct, that comprises or essentially    consists of one or more derivatives according to any of aspects T-1    to T-14, and optionally further comprises one or more other groups,    residues, moieties or binding units, optionally linked via one or    more linkers.-   Aspect T-16: Compound or construct according to aspect 1-15, in    which said one or more other groups, residues, moieties or binding    units are amino acid sequences.-   Aspect T-17: Compound or construct according to aspect T-16, in    which said one or more linkers, if present, are one or more amino    acid sequences.-   Aspect T-18: Nucleic acid encoding a compound or construct according    to aspect T-16 or T-17.-   Aspect T-19: Composition, comprising at least one derivative to any    of aspects T-1 to T-14, compound or construct according to any of    aspects T-15 to T-17, or nucleic acid or nucleotide sequence    according to aspect T-18.

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference, in particular for theteaching that is referenced hereinabove.

The invention will now be further described by means of the followingnon-limiting figures, experimental part and preferred aspects:

Figures:

FIG. 1: SDS_PAGE—The results show that 7 out of the 8 nanobodies werebound on the beads after the procedure, only nanobody 99D3 was notdetected. From the seven binding nanobodies six were able to precipitateRAGE out of the solution. 99C6 was not able to precipitate RAGE. 99B5,108D3 and 108E3 were most efficient, since they seem to precipitatealmost all of the applied RAGE (most right).

FIG. 2: Cell viability after treatment with anti-RAGE clones in theabsence and presence of TTR oligomers.

EXPERIMENTAL PART Example 1 Generation and Characterization of Anti-RAGENanobodies Example 1.1 Animal Immunizations

Two llamas 100 and 101 were immunized, according to standard protocols,with 6 boosts of a cocktail containing 1145-RAGE, i.e. the extracellularpart of recombinant human RAGE fused to human Fc (R&D Systems Cat No1145-RG). This recombinant protein was obtained from a DNA sequenceencoding the extracellular domain of recombinant human RAGE (Pro100-Lys330), fused to human Fc region of human IgG1 at the C-terminus. Bloodwas collected from these animals 4 and 8 days after boost 6.

Example 1.2 Library Construction

Peripheral blood mononuclear cells were prepared from blood samplesusing Ficoll-Hypaque according to the manufacturer's instructions. Next,total RNA were extracted from these cells and lymph node tissue and usedas starting material for RT-PCR to amplify Nanobody encoding genefragments. These fragments were cloned into phagemid vector pAX50. Phagewere prepared according to standard methods (see for example the priorart and applications filed by applicant cited herein) resulting in phagelibraries 100 and 101.

Example 1.3 Selections

Phage libraries 100 and 101 were used for selections on extracellularpart of recombinant human RAGE fused to human Fc (R&D Systems Cat No1145-RG; hereinafter also “rhRG”). The rhRG was coated independently at5 ug/ml (wherein hereinafter and before ug=μg), 0.5 ug/ml and 0 ug/ml(control) on Maxisorp 96 well microtiter plates (Nunc). To minimize thenumber of phage binding to the Fc-portion of rhRG the phage werepre-incubated with 100 ug/ml human IgG (SIGMA, Cat No 14506, Lot04710635). Following incubation with the phage libraries and extensivewashing, bound phages were eluted with either 1 mg/ml trypsin (in PBS)or 100 ug/ml AGE-BSA (US Biological; A0909-75; Prepared by reacting BSAwith glycoaldehyde under sterile conditions). The eluted phage from the5 ug/ml rhRG coat and library 101 were further amplified and applied ina second round of selection on 5 ug/ml immobilized rhRG and eluted witheither 1 mg/ml trypsin (in PBS) or 100 ug/ml AGE-BSA (US Biological;A0909-75; Prepared by reacting BSA with glycoaldehyde under sterileconditions). Individual colonies of E. coli TG1 infected with theobtained eluted phage pools were grown and i) induced for new phageproduction and ii) induced with IPTG for Nanobody expression andextraction (periplasmic extracts) according to standard methods (see forexample the prior art and applications filed by applicant cited herein).

Example 1.4 Screening

In order to determine binding specificity to rhRG, the clones weretested in an ELISA binding assay setup, using the monoclonal nanobodypools. Nanobody binding to immobilized rhRG were tested. Shortly, 100 ul5 ug/ml rhRG was coated on Maxisorp ELISA plates (Nuns) in PBS overnight at 4 degree Celcius. After washing the plate was blocked with 4%MPBS for 30 minutes hour on shaker. 10 ul of periplasmic extractcontaining nanobody was added in 100 ul 2% Marvell/PBS and incubate for1 hour on shaker. Nanobody was detected with 100 ul 1:5000Mouse-anti-MycTag (9E10) in 2% Marvell/PBS and with 100 ul 1:5000 DAMPOin 2% Marvell/PBS for 1 hour on shaker. After washing PO was detectedwith 100 ul OPD and stopped with 50 ul H2SO4. Quantification wasperformed at 490 nm in a plate reader. The results (raw data not shown)indicate that 103 out of 144 (both 1^(st) and second round) clonespicked were positive for binding to rhRG.

Example 1.5 Dose-Dependent Binding of Selected Anti-RAGE Nanobodies

Maxisorp plate was coated with 100 ul 2 ug/ml of rhRG in PBS 0/N at 4 C.After washing the plate was blocked with 4% MPBS for 30 minutes onshaker. Nanobodies 99B5, 99C6, 99D3, 108D3, 108D11, 108E3, 108G10,108G12, and 49C5* (4-1000 nM) were added in 100 ul 2% Marvell/PBS andincubated for 1 hour on shaker. After washing the nanobodies weredetected with 100 ul 1:5000 Mouse-anti-MycTag (9E10) in 2% Marvell/PBSfor 1 hour on shaker. After washing the anti-myc was detected with 100ul 1:5000 DAMPO in 2% Marvell/PBS for 1 hour on shaker. Peroxidase wasdetected with 100 ul OPD and stopped with 50 ul H2SO4. Quantificationwas performed at 490 nm in a plate reader. The results show thatnanobody 49C5 (negative control), 99C6 and 99D3 do not bind RAGE, butthe other nanobodies 99B5, 108D3, 108D11, 108E3, 108G10, 108G12 do bindRAGE in a dose dependent matter.

*49C5: Negative control antibody—generation and characterizationpublished in WO2008/074867 (see also herein SEQ ID NO: 337:

-   EVQLVESGGGLVQAGGSLGLSCVASGRTFVSGGMGWFRQAPGKEREFVASIVWIGG    RTYYADSVKGRFTISGDNAKSTVTLQMNSLKPEDTATYYCADGDLATGTYDYWGQ GTQVTVSS)

Example 1.6 Immunoprecipitation with Selected Anti-RAGE Nanobodies

In order to test the binding of anti-RAGE clones further anImmunoprecipitation experiment was performed. In short, 100 ul of Talonslurry (50 ul beads) was washed carefully with lysis buffer (RIPAwithout EDTA) (20 mM tris pH7.4; 150 mM NaCl; 0.5% TritonX100; 0.1%SDS). 10 ug of VHHs was added to the beads in 200 ul of lysis buffer andincubate for 30 min hoh. The beads were washed once with lysis bufferand 1 ug of rhRG was added to the beads (100 ul) and incubated for 1hour. The beads were spinned down and supernatant was removed. The beadswere washed twice. Subsequently the beads were boiled for 5 min insample buffer and the proteins were separated on a SDS PAGE by gelelectrophoresis (FIG. 1).

The high molecular weight part was detected with mouse-anti-RAGEantibody (R&D systems) and the lower part was detected with mouseanti-myc.

The results show that 7 out of the 8 nanobodies were bound on the beadsafter the procedure, only nanobody 99D3 was not detected. From the sevenbinding nanobodies six were able to precipitate RAGE out of thesolution. 99C6 was not able to precipitate RAGE. 99B5, 108D3 and 108E3were most efficient, since they seem to precipitate almost all of theapplied RAGE (most right).

Example 1.7 Functional Assay with Selected Anti-RAGE Nanobodies

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominantneurodegenerative disorder related to the systemic deposition of mutatedtransthyretin (TTR) amyloid fibrils, particularly in peripheral nervoussystem (PNS). The toxic nature of TTR non-fibrillar aggregates, and notmature TTR fibrils, was evidenced by their ability to induce theexpression of oxidative stress and inflammation-related molecules inneuronal cells, driving them into apoptotic pathways. How these TTRaggregates exert their effects is debatable; interaction with Cellularreceptors, namely the receptor for advanced glycation endproducts is aprobable candidate mechanism. The pathology and the molecular signalingmechanisms responsible for neurodegeneration in FAP is not clearlyestablished (Inflammation and Apoptotic Pathways in the PeripheralNervous System Related to Protein Misfolding, Springer, ISBN978-0-387-70830-0 (Online), 2007). Previously it was described thatinteraction of TTR aggregates with RAGE contributes to cellular stressand toxicity in FAP (Sousa et al., The Journal of Neuroscience, Oct. 1,2001, 21(19):7576-7586). In the present study we evaluate the ability ofdifferent RAGE antibodies to inhibit toxicity induced by TTR aggregatesin cells.

TTR Oligomers production Production of TTR oligomers were achieved asdescribed (Teixeira et al, J. Biol. Chem., Vol. 281, Issue 31,21998-22003, Aug. 4, 2006).

Cell assays: SH-SY5Y (human neuroblastoma cell line) were propagated in25 cm² flasks in monolayers and maintained at 37° C. in a humidifiedatmosphere of 95% and 5% CO₂. Cells were grown in MEM:F12 supplementedwith 15% FBS, 1% MEM Non-essential amino acid solution, 100 U/mLPenicillin/Streptomycin, 2 mM Glutamine. Cells were plated in sixchamber lab-teks. Cells were preincubated for 3 h in complete media with3.75 ug/mL of the indicated nanobodies/antibodies to be tested (TableC-1). Following this the cells were incubated for 12 h in serum freeassay media with 1.88 ug/mL of antibody and 2 uM of TTR oligomers.

TABLE C-1 nanobodies/antibodies to be tested Clone 108E3 α RAGE 108G12 αRAGE 108D3 α RAGE 99B5 α RAGE 99C8 α SR-A1 (negative control) PS2-8 MEK(negative control)

Cell Viability assay: In order to analyze cell viability and accessedcell dead we performed LIVE/DEAD viability/cytotoxicity assay (MolecularProbes), this method is based in the capacity of discriminate betweenlive and dead cells. Live cells are distinguished by the presence ofubiquitous intracellular esterase activity, determined by the enzymaticconversion of the virtually nonfluorescent cell-permeant calcein AM tothe intensely fluorescent calcein. The polyanionic dye calcein is wellretained within live cells, producing an intense uniform greenfluorescence in live cells. Ethidium homodimer (EthD-1) enters cellswith damaged membranes and undergoes a 40-fold enhancement offluorescence upon binding to nucleic acids, thereby producing a brightred fluorescence in dead cells. EthD-1 is excluded by the intact plasmamembrane of live cells. Cultures were incubated with 2 uM EthD-1 and 4uM of Calcein AM in Phosphate Buffer Saline (PBS) for 30 min at roomtemperature. The labeled cells were then viewed under a fluorescentmicroscope in order to quantify living and dead cells.

The presence of oTTR is expected to induce cell dead, however, treatmentof cells with these antibodies 99B5 and 108G12 in the presence of oTTRdidn't induce an increase of cell dead. Treatment with anti-RAGE clones99B5, 108G12 suppressed the effects of oTTR induced cytotoxicity.Treatment with clones PS2-8, 99C8, 108D3 and 108E3 didn't suppress oTTRinduced cell dead (FIG. 2).

Classification of Amyloid (See e.g. Wikipedia)

Approximately 25 different proteins are known that can form amyloid inhumans. Most of them are constituents of the plasma.

Different amyloidoses can be systemic (affecting many different organsystems) or organ-specific. Some are inherited, due to mutations in theprecursor protein. Other forms are due to different diseases causingoverabundant or abnormal protein production-such as with over:production of immunoglobulin light chains in multiple myeloma (termed ALamyloid), or with continuous overproduction of acute phase proteins inchronic inflammation (which can lead to AA amyloid).

There are at least 15 biologically distinct forms of amyloid, some moreclinically significant than others. Following is a brief description ofthe more common types of amyloid:

Abb. Amyloid type Description AL amyloid light Contains immunoglobulinlight-chains (λ,κ) chain derived from plasma cells AA amyloidNon-immunoglobulin protein made in the liver associated Aβ β amyloidFound in Alzheimer disease brain lesions ATTR Transthyretin A mutantform of a normal serum protein that is deposited in the geneticallydetermined familial amyloid polyneuropathies. TTR is also deposited inthe heart in senile systemic amyloidosis.^([5]) Aβ₂ m β₂ Not to beconfused with Aβ, β₂m is a normal microglobulin serum protein, part ofmajor histocompatability complex (MHC) Class 1 molecules. Can occur inlong term haemodialysis. PrP Prion related In Prion diseases, misfoldedprion proteins protein deposit in tissues and resemble amyloid proteins.

OMIM includes the following (The Mendelian Inheritance in Man project isa database that catalogues all the known diseases with a geneticcomponent, and—when possible—links them to the relevant genes in thehuman genome and provides references for further research and tools forgenomic analysis of a catalogued gene. The online version is calledOnline Mendelian Inheritance in Man (OMIM), which can be accessed withthe Entrez database searcher of the National Library of Medicine and ispart of the NCBI project Education):

OMIM Gene Name Number 176300 TTR Senile systemic amyloidosis (type 1)105120 GSN Finnish type amyloidosis (type 5) 105150 CST3 Cerebralamyloid angiopathy, Icelandic (type 6) type 105210 TTR Leptomeningealamyloidosis (type 7) 105200 APOA1, Familial visceral amyloidosis (type8) FGA, LYZ 105250 OSMR Primary cutaneous amyloidosis (type 9) 176500ITM2B Cerebral amyloid angiopathy, British type — 609065, APP Dutchtype/Italian type/Iowa type — 605714

The amyloidoses can be classified as systemic or localised, primary orsecondary, or according to which type of amyloid is deposited. Byconvention, a combination of these approaches are used clinically.

Systemic amyloidoses are those which affect more than one body organ orsystem. Localised amyloidoses affect only one body organ or tissue type.

Primary amyloidoses arise from a disease with disordered immune cellfunction such as multiple myeloma and other immunocyte dyscrasias.

Secondary (reactive) amyloidoses are those occurring as a complicationof some other chronic inflammatory or tissue destructive disease.

The different types of amyloid deposited (see table, above) may beeither primary or secondary, systemic or localised.

The common systemic amyloidoses:

Amyloid Category type Precursor protein Amyloidosis Systemic ALImmunoglobin AL amyloidosis (primary acquired light chains amyloidosis)(Bence Jones protein) Systemic AA SAA AA amyloidosis (secondaryhereditary amyloidosis) Systemic Aβ₂ m β₂ microglobulin Haemodialysisassociated hereditary Systemic AA SAA Familial mediterranean feverhereditary Systemic ATTR transthyretin Familial amyloidotic hereditarypolyneuropathies Systemic ATTR transthyretin Systemic senile amyloidosishereditary

Example 1.8 Sequencing of Selected Nanobodies

See SEQ ID NO: 308 to 315

Example 2 Generation and Characterization of Anti-Lox-1 NanobodiesExample 2.1 Animal Immunizations

Two llamas 100 and 101 were immunized, according to standard protocols,with 6 boosts of a cocktail containing 1798-LX, i.e. the extracellularpart of recombinant human Lox-1 fused to polyhistidine tag (R&D Systems1, cat nr. 1798-LX). This recombinant protein was obtained from a DNAsequence encoding the extracellular domain of recombinant human Lox-1(Ser 61—Gln273), fused to an N-terminal polyhistidine tag (9histidines). Blood was collected from these animals 4 and 8 days afterboost 6.

Example 2.2 Library Construction

Peripheral blood mononuclear cells were prepared from blood samplesusing Ficoll-Hypaque according to the manufacturer's instructions, Next,total RNA were extracted from these cells and lymph node tissue and usedas starting material for RT-PCR to amplify Nanobody encoding genefragments. These fragments were cloned into phagemid vector pAX50. Phagewere prepared according to standard methods (see for example the priorart and applications filed by applicant cited herein) resulting in phagelibraries 100 and 101.

Example 2.3 Selections

Phage libraries 100 and 101 were used for selections on extracellularpart of recombinant human Lox-1 fused to polyhistidine tag (R&D Systems1, cat nr. 1798-LX), hereinafter also “rhLox-1”). The rhLox-1 was coatedindependently at 5 ug/ml (wherein hereinafter and before ug=μg), 0.5ug/ml and 0 ug/ml (control) on Maxisorp 96 well microtiter plates(Nunc). Following incubation with the phage libraries and extensivewashing, bound phages were eluted with either 1 mg/ml trypsin (in PBS)or 100 ug/ml AGE-BSA (US Biological; A0909-75; Prepared by reacting BSAwith glycoaldehyde under sterile conditions). The eluted phage from the5 ug/ml rhLox-1 coat and library 101 and 100 were further amplified andapplied in a second round of selection on 5 ug/ml immobilized rhLox-1and eluted with either 1 mg/ml trypsin (in PBS) or 100 ug/ml AGE-BSA (USBiological; A0909-75; Prepared by reacting BSA with glycoaldehyde understerile conditions). Individual colonies of E. coli TG1 infected withthe obtained eluted phage pools were grown and i) induced for new phageproduction and ii) induced with IPTG for Nanobody expression andextraction (periplasmic extracts) according to standard methods (see forexample the prior art and applications filed by applicant cited herein).

Example 2.4 Screening

In order to determine binding specificity to rhLox-1, the clones weretested in an ELISA binding assay setup, using the monoclonal nanobodypools. Nanobody binding to immobilized rhLox-1 were tested. Shortly, 100ul 5 ug/ml rhLox-1 was coated on Maxisorp ELISA plates (Nunc) in PBSover night at 4 degree Celcius. After washing the plate was blocked with4% MPBS for 30 minutes hour on shaker. 10 ul of periplasmic extractcontaining nanobody was added in 100 ul 2% Marvell/PBS and incubate for1 hour on shaker.

Nanobody was detected with 100 ul 1:5000 Mouse-anti-MycTag (9E10) in 2%Marvell/PBS and with 100 ul 1:5000 DAMPO in 2% Marvell/PBS for 1 hour onshaker. After washing PO was detected with 100 ul OPD and stopped with50 ul H2SO4. Quantification was performed at 490 nm in a plate reader.The results (raw data not shown) indicate that 31 out of 93 clonespicked were positive for binding to rhLox-1.

Example 2.5 Dose-Dependent Binding of Selected Anti-Lox-1 Nanobodies

Maxisorp plate was coated with 100 ul 2 ug/ml of rhLox-1 in PBS O/N at 4C. After washing the plate was blocked with 4% MPBS for 30 minutes onshaker. Nanobodies 98E7, 98D8, 98B9, 98E9, 98A10, 98F12, 105B5, 105F7,105A8, 105B8 and irrelevant nanobody 49C5 (against alphaFcgRI) (1-1000nM) were added in 100 ul 2% Marvell/PBS and incubated for 1 hour onshaker. After washing the nanobodies were detected with 100 ul 1:5000Mouse-anti-MycTag (9E10) in 2% Marvell/PBS for 1 hour on shaker. Afterwashing the anti-myc was detected with 100 ul 1:5000 DAMPO in 2%Marvell/PBS for 1 hour on shaker. Peroxidase was detected with 100 ulOPD and stopped with 50 ul H2SO4. Quantification was performed at 490 nmin a plate reader. The results show that nanobody 105B5 and 49C5(negative control) does not bind rhLox-1, but the other nanobodies 98E7,98D8, 98B9, 98E9, 98A10, 98F12, 105F7, 105A8, 105B8 do bind rhLox-1 in adose dependent matter.

There are first indications that some of the anti-Lox-1 nanobodies canbe used in immunohistochemistry.

Example 2.6 Sequencing of Selected Nanobodies

See SEQ ID NO: 316 to 326

Example 3 Generation and Characterization of Anti-SR-A1 NanobodiesExample 3.1 Animal Immunizations

Two llamas 164 and 165 were immunized, according to standard protocols,with 6 boosts of a cocktail containing 2708-MS, i.e. the extracellularpart of recombinant human SR-A1 fused to polyhistidine tag (R&D SystemsCat No 2708-MS). This recombinant protein was obtained from a DNAsequence encoding the extracellular domain of recombinant human SR-A1(Lys 77-Leu 451), fused to a N-terminal polyhistidine tag (9histidines). Blood was collected from these animals 4 and 8 days afterboost 6.

Example 3.2 Library Construction

Peripheral blood mononuclear cells were prepared from blood samplesusing Ficoll-Hypaque according to the manufacturer's instructions. Next,total RNA were extracted from these cells and lymph node tissue and usedas starting material for RT-PCR to amplify Nanobody encoding genefragments. These fragments were cloned into phagemid vector pAX50. Phagewere prepared according to standard methods (see for example the priorart and applications filed by applicant cited herein) resulting in phagelibraries 164 and 165.

Example 3.3 Selections

Phage libraries 164 and 165 were used for selections on extracellularpart of recombinant human SR-A1 fused to polyhistidine tag (R&D SystemsCat No 2708-MS) hereinafter also “rhSRA-1”). The rhSRA-1 was coatedindependently at 5 ug/ml (wherein hereinafter and before ug=μg), 0.5ug/ml and 0 ug/ml (control) on Maxisorp 96 well microtiter plates(Nunc). Following incubation with the phage libraries and extensivewashing, bound phages were eluted with either 1 mg/ml trypsin (in PBS)or 100 ug/ml AGE-BSA (US Biological; A0909-75; Prepared, by reacting BSAwith glycoaldehyde under sterile conditions). The eluted phage from the5 ug/ml rhSRA-1 coat and library 164 and 165 were further amplified andapplied in a second round of selection on 5 ug/ml immobilized rhSRA-1and eluted with either 1 mg/ml trypsin (in PBS) or 100 ug/ml AGE-BSA (USBiological; A0909-75; Prepared by reacting BSA with glycoaldehyde understerile conditions). Individual colonies of E. coli TG1 infected withthe obtained eluted phage pools were grown and i) induced for new phageproduction and ii) induced with IPTG for Nanobody expression andextraction (periplasmic extracts) according to standard methods (see forexample the prior art and applications filed by applicant cited herein).

Example 3.4 Screening

In order to determine binding specificity to rhSRA-1, the clones weretested in an ELISA binding assay setup, using the monoclonal nanobodypools. Nanobody binding to immobilized rhSRA-1 were tested. Shortly, 100ul 5 ug/ml rhSRA-1 was coated OD Maxisorp ELISA plates (Nunc) in PBSover night at 4 degree Celcius. After washing the plate was blocked with4% MPBS for 30 minutes hour on shaker. 10 ul of periplasmic extractcontaining nanobody was added in 100 ul 2% Marvell/PBS and incubate for1 hour on shaker. Nanobody was detected with 100 ul 1:5000Mouse-anti-MycTag (9E10) in 2% Marvell/PBS and with 100 ul 1:5000 DAMPOin 2% Marvell/PBS for 1 hour on shaker. After washing PO was detectedwith 100 ul OPD and stopped with 50 ul H2SO4. Quantification wasperformed at 490 nm in a plate reader. The results (raw data not shown)indicate that 56 out of 78 clones picked were positive for binding torhSRA-1.

Example 3.5 Dose-Dependent Binding of Selected Anti-SR-A1 Nanobodies

Maxisorp plate was coated with 100 ul 2 ug/ml of rhSRA-1 in PBS 0/N at 4C. After washing the plate was blocked with 4% MPBS for 30 minutes onshaker. Nanobodies 99D11, 99C9, 105A11, 105F12, 105G11, 105D9, 99C8 andirrelevant nanobody 49C5 (against alphaFcgRI) (1-1000 nM) were added in100 al 2% Marvell/PBS and incubated for 1 hour on shaker. After washingthe nanobodies were detected with 100 ul 1:5000 Mouse-anti-MycTag (9E10)in 2% Marvell/PBS for 1 hour on shaker. After washing the anti-myc wasdetected with 100 ul 1:5000 DAMPO in 2% Marvell/PBS for 1 hour onshaker. Peroxidase was detected with 100 ul OPD and stopped with 50 ulH2SO4. Quantification was performed at 490 nm in a plate reader. Theresults show that nanobody 99D11 and 49C5 (negative control) does notbind rhSRA-1, but the other nanobodies 99C9, 105A11, 105F12, 105G11,105D9, 99C8 do bind rhSRA-1 in a dose dependent matter.

Example 3.6 Sequencing of Selected Nanobodies

See SEQ ID NO: 327 to 333

The invention claimed is:
 1. A single domain antibody that specificallybinds to human RAGE comprising the amino acid sequence of SEQ ID NO:334, wherein an amino acid sequence of the single domain antibodycomprises 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively) in thestructure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein CDR1 comprises theamino acid sequence of SEQ ID NO: 152, CDR2 comprises the amino acidsequence of 204, and CDR3 comprises the amino acid sequence of SEQ IDNO:
 256. 2. The single domain antibody according to claim 1, wherein theamino acid sequence of the single domain antibody comprises the aminoacid sequence of SEQ ID NO:
 308. 3. The single domain antibody of claim1, wherein the FR1, FR2, FR3, and FR4 regions comprise an amino acidsequence that is 80% identical to the amino acid sequence of the FR1,FR2, FR3, and FR4 regions of SEQ ID NO:
 308. 4. The single domainantibody of claim 3, wherein the FR1, FR2, FR3, and FR4 regions comprisean amino acid sequence that is 90% identical to the amino acid sequenceof the FR1, FR2, FR3, and FR4 regions of SEQ ID NO:
 308. 5. The singledomain antibody according to claim 1 that can prevent the increase ofTTR oligomers induced cell death of cells, wherein said cells expressfunctional human RAGE.
 6. The single domain antibody of claim 5, whereinthe cells are human neuroblastomas such as SH-SY5Y.
 7. The single domainantibody according to claim 1, wherein the single domain antibody is aVHH, a humanized VHH or a camelized VH.
 8. A single domain antibody oran antibody that binds the same epitope of human RAGE comprising theamino acid sequence of SEQ ID NO: 334 as a polypeptide comprising theamino acid sequence of SEQ ID NO:
 308. 9. The single domain antibody orantibody according to claim 8, wherein the ability of said single domainantibody or antibody to bind to the same epitope as a polypeptidecomprising the amino acid sequence of SEQ ID NO: 308 is detected byusing surface plasmon resonance or by using an ELISA assay.
 10. A methodfor the treatment of amyloidosis comprising administering, to a subjectin need thereof, a pharmaceutically active amount of the single domainantibody of claim
 1. 11. The method for the treatment of amyloidosisaccording to claim 10, wherein said amyloidosis is in Alzheimer'sDisease or Familial amyloidotic polyneuropathy (FAP).